OmpA and ASP14 in vaccine compositions and as diagnostic targets

ABSTRACT

Anaplasma phagocytophilum  surface proteins Asp14 and OmpA and homologous genes from Anaplasmatacaea family members are used in compositions suitable for vaccines to treat or prevent infections caused by tick-born bacteria of the Anaplasmatacaea family. Asp14 and/or OmpA proteins or peptide fragments may be used in combination with other Anaplasmatacaea surface proteins to elicit an immune response. Furthermore, antibodies to Asp14 and/or OmpA proteins can be used in diagnostic methods to determine whether an individual has contracted an Anaplasmatacaea infection. Because of the conserved invasin domains in the surface proteins, a wide range of Anaplasmatacaea infections may be diagnosed, treated or prevented using compositions of the invention.

PRIORITY

This application claims the benefit of U.S. application 61/665,223,filed Jun. 27, 2012, and U.S. application 61/698,979, filed Sep. 10,2012. These applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention generally relates to a vaccine and diagnostic foranaplasmosis in animals and humans. In particular, the inventionprovides Anaplasma phagocytophilum outer surface protein A (OmpA)epitopes and/or Anaplasma phagocytophilum surface protein 14 (Asp14)epitopes.

SEQUENCE LISTING

This document incorporates by reference an electronic sequence listingtext file, which was electronically submitted along with this document.The text file is named 02940826TA_ST25.txt, is 87 kilobytes, and wascreated on Jun. 17, 2013.

BACKGROUND OF THE INVENTION

Anaplasma phagocytophilum (Aph) is a tick-transmitted obligateintracellular bacterium of the family Anaplasmataceae that can infecthumans, livestock, companion animals and wild animals. In addition toAph, the Anaplasmataceae family members include Anaplasma marginale,Anaplasma platys, Ehrlichia chaffeensis, Ehrlichia canis, and Ehrlichiaruminatium, among others, and all of these cause similar infectionsknown collectively as ehrlichiosis. When humans contract an Aphinfection it is more specifically known as human granulocyticanaplasmosis (HGA). An emerging and potentially fatal disease, HGA istransmitted by the same vectors that transmit Lyme Disease, primarilyticks and deer, but other animal and human hosts can complete the vectorcycle and therefore extend the spread of disease. Since HGA became areportable disease in the U.S. in 1999, the number of cases has risenannually, reaching 1,761 in 2010. Since diagnostic tools for HGA arelacking, this number of actual cases is likely to be much higher. HGA isincreasingly recognized in Europe and Asia, and Aph infection is now themost widespread tick-transmitted disease of animals in Europe.

As the name implies, an obligate intracellular bacterium must enter atarget cell in its human or animal host to survive, replicate, and moveto the next host. When an Anaplasma spp. or Ehrlichia spp. infected tickbites a subject, the bacteria is transferred from the tick salivaryglands into the tissues of the host or into the bloodstream where theybind to the surface of host cells and are taken up into vacuoles thatform around each bacterium. A resident bacterium prevents the vacuolefrom merging with lysosomes. In doing so, the bacterium converts thecell into a protective niche that favors bacterial survival and remainsin circulation to enable it to complete its zoonotic cycle. While thehallmark of these diseases is Aph colonization of neutrophils, otherpathological findings can include leukopenia, thrombocytopenia, andelevated serum transaminase levels. Anaplasmosis is marked by fever andincreased susceptibility to potentially fatal opportunistic infections.

Infected neutrophils in a host can be ingested in a second tickbite/bloodmeal. In this manner the disease may be transferred to anothersubject bitten subsequently. However, HGA can also be transmittedperinatally or by blood transfusion, and possibly nosocomially. Blockinginfection of neutrophils could conceivably prevent all of these types ofdissemination of Aph infection and the increased risk of opportunisticinfections that can accompany the disease. Furthermore, targeting Aphproteins that are conserved among related Anaplasmataceae family membersmight also reduce or block transmission of disease caused by relatedAnaplasmataceae family members.

In addition to neutrophils, Aph has been detected in the microvascularendothelium of heart and liver in experimentally infected severecombined immunodeficiency mice. Promyelocytic and endothelial cell linesare useful in vitro models for studying Aph-host cell interactions. Whennaïve neutrophils or HL-60 cells are overlaid on Aph-infectedendothelial cells, the bacterium rapidly transmigrates into the myeloidcells. These observations demonstrated that Aph infects endothelialcells in vivo and suggested that the bacterium may transmigrate betweenendothelial cells and neutrophils during the course of mammalianinfection. Thus, targeting the Aph invasins that mediate infection ofendothelial cells may prevent movement of the bacteria into themicrovasculature of heart and liver in an affected subject by blockingthe mechanism of endothelial cell-to-neutrophil transfer.

The Aph genome has now been sequenced and annotated. The annotated Aphgenome (HZ strain, isolated from a human patient) can be found on thewebsite at cmr.jcvi.org, and more specifically on the page found atcmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?org_search=&org=gaph.Another website is the page found at ncbi.nlm.nih.gov, more specificallyon the page found atncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode-Info&id=948&lvl=3&keep=1&srchmode=1&unlock.This information has promoted some progress regarding diagnosis ofanaplasmosis and protecting subjects from infection. U.S. Pat. No.7,906,296 B2 to Beall et al teaches that Anaplasma platys (Apl),formerly known as Ehrlichia platys, causes tick-born anaplasmosis indogs. Beall further teaches the uses of Apl polypeptides. The Aplsequences were amplified from a blood sample of a dog known to beinfected with Apl in order to develop methods for detecting the presenceof antibodies to Apl and/or Aph in dog sera. Beall also teaches thatpeptides including Apl P44, encoding a major surface protein, might beused to elicit an immune response in vivo and confer resistance toanaplasmosis caused by Apl or Aph. U.S. Pat. No. 8,158,370 B2 to Liu elal. teaches that polypeptide sequences amplified from either Apl or Aphcan be used in diagnostic assays, or to induce an immune reaction thatmight confer protection from Apl or Aph infection. The Aph sequencesused by Liu were derived from APH_0915, encoding a protein of unknownfunction. More recently, Liu et al., in U.S. Pat. No. 8,303,959 and US2013/0064842, teaches the use of four strain variants of Aph P44 surfaceproteins to diagnosis and protect against anaplasmosis.

Despite these teachings, there are currently no human or animal vaccinesagainst anaplasmosis caused by any of the Anaplasmataceae familymembers. Antibiotic treatments with doxycycline or tetracycline can beeffective, but tools for rapid and definitive diagnosis in any speciesother than dogs are lacking. The current gold standard serologic testfor diagnosis of anaplasmosis in humans is indirect immunofluorescenceassays (IFA) performed as timed pairs over a period of a few weeks, onlyavailable in specialized reference laboratories. This assay measuresnon-specific increases in IgM and IgG antibody levels. However, IgMantibodies, which usually rise at the same time as IgG near the end ofthe first week of illness and remain elevated for months or longer, areeven less specific than IgG antibodies and more likely to result in afalse positive. Serologic tests based on enzyme immunoassay (EIA)technology are available from some commercial laboratories. However, EIAtests are qualitative rather than quantitative, meaning they onlyprovide a positive/negative result, and are less useful to measurechanges in antibody titers between paired specimens. Furthermore, someEIA assays rely on the evaluation of IgM antibody alone, which again mayhave a higher frequency of false positive results. Between 5-10% ofcurrently healthy people in some areas may have elevated antibody titersdue to past exposure to Aph or related family members. If only onesample is tested it can be difficult to interpret. A four-fold rise inantibody titer is needed to achieve significance in paired samples takenweeks apart.

Therefore, the need remains for compositions and methods to rapidly andaccurately diagnosis new cases and to provide adequate vaccinationagainst Anaplasmataceae infections that cause anaplasmosis and HGA.

SUMMARY

An embodiment of the invention is a composition including one or moreisolated polypeptides, wherein at least one of said one or morepolypeptides is or includes SEQ ID NO:03 or SEQ ID NO:06. The one ormore polypeptides may be linked to an amino acid spacer, an amino acidlinker, a signal sequence, a stop transfer sequence, a transmembranedomain, a protein purification ligand, a heterologous protein, or one ormore additional polypeptides comprising SEQ ID NO:01, 02, 03, 04, 05 or06, or a combination thereof. An exemplary embodiment is one or morepolypeptides linked to a protein purification ligand, and proteinpurification ligands are a peptide encoding a histidine tag.

Another embodiment of the invention comprises one or more polypeptidesselected from the group consisting of SEQ ID NO:01, SEQ ID NO:02, andSEQ ID NO:03. Another embodiment of the invention comprises one or morepolypeptides selected from the group consisting of SEQ ID NO:04, SEQ IDNO:05, and SEQ ID NO:06.

Another embodiment of the invention is a method of protecting a subjectfrom acquiring a zoonotic disease and/or treating a zoonotic disease ina subject by the step of administering a composition including one ormore isolated polypeptides, wherein at least one of said one or morepolypeptides is or includes SEQ ID NO:03 or SEQ ID NO:06. The one ormore polypeptides may be linked to an amino acid spacer, an amino acidlinker, a signal sequence, a stop transfer sequence, a transmembranedomain, a protein purification ligand, a heterologous protein, or one ormore additional polypeptides comprising SEQ ID NO:01, 02, 03, 04, 05 or06; or a combination thereof. The zoonotic disease may be one caused byan obligate intracellular Anaplasmataceae bacterium selected from thegroup consisting of Anaplasma phagocytophilum, Anaplasma marginale,Anaplasma platys, Ehrlichia chaffeensis, Ehrlichia canis, and Ehrliciaruminatium. When the subject is a human, and the zoonotic disease may behuman granulocytic anaplasmosis (HGA). When the subject is an animal,the zoonotic disease may be anaplasmosis.

Another embodiment of the invention is a method of detecting antibodiesthat specifically bind an Anaplasmataceae polypeptide in a test sample.The method may include the steps of contacting a test sample, underconditions that allow polypeptide-antibody complexes to form, with acomposition that includes at least one or more polypeptides encoding allor a portion of SEQ ID NO:01 or SEQ ID NO:04, and detecting saidpolypeptide-antibody complexes, wherein the detection is an indicationthat antibodies specific for Anaplasmataceae Asp14 or OmpA are presentin the test sample. The method may be an assay selected from the groupconsisting of an immunoblot and an enzyme-linked immunosorbent assay(ELISA). An exemplary embodiment of this methodology may include usingat least one polypeptide which is or includes SEQ ID NO:03 or SEQ IDNO:06 in the assay, whereby infection with obligate intracellularAnaplasmataceae is determined from a serum sample exhibiting antibodybinding with the at least one polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and B. Timeline of Aph infection cycle and differentialtranscription profiling of OMP candidate genes throughout the Aphinfection cycle.

FIG. 1C-E. Differential transcription profiling of OMP candidate genesthroughout the Aph infection cycle.

FIG. 2A-D. Differential expression analyses of ompA and asp14 during Aphinvasion of HL-60 and RF/6A cells, during Aph binding to PSGL-1 CHOcells, and during transmission feeding of Aph infected I. scapularisticks.

FIG. 3A-G. Aph expresses OmpA and Asp14 during infection of HL-60 cellsand during murine and human infection.

FIGS. 4A and B. Trypsin treatment abolishes detection of Aph surfaceproteins and surface proteins Asp14 and OmpA are detected in Aph DCorganisms.

FIG. 5A-D. Anti-OmpA does not disrupt bacterial cellular adherence orbacterial interaction with PSGL-1, but does partially neutralize Aphinfection of HL-60 cells.

FIGS. 6A and B. Alignment of OmpA (SEQ ID NO:04) with Anaplasma andEhrlichia species homologs AM854 (SEQ ID NO: 31), ACHIS_00486 (SEQ IDNO:33), ECH_0462 (SEQ ID NO:39), Ecaj_0563 (SEQ ID NO:45), and Erum 5620(SEQ ID NO:51) with regions of identity and similarity shaded, andpredicted 3D structure with extracellular loop and helix are indicatedby arrows.

FIGS. 7A and B. Pretreatment of Aph with anti-OmpA reduces infection ofHL-60 cells.

FIG. 8A-D. Model for how Aph OmpA interacts with its receptor to promoteinfection of host cells

FIG. 9A-D. Pretreatment of Aph with anti-Asp14 reduces infection ofHL-60 cells.

FIG. 10A-D. Asp14 residues 101-124 are required to competitively inhibitAph infection of mammalian host cells.

FIG. 11. An alignment of Asp14 residues 101-115, which constitute aconserved domain among homologs from Anaplasma and Ehrlichia speciesAsp14 (SEQ ID NO:01), AM936 (SEQ ID NO:13), ACHIS_00403 (SEQ ID NO:15),ECH_0377 (SEQ ID NO:19), Ecaj_0636 (SEQ ID NO:23), and Erum6320 (SEQ IDNO:27) with regions of identity and similarity shaded.

FIGS. 12A and B. Recombinant forms of Asp14 and OmpA cooperatively blockAph infection of HL-60 cells, either as full-length proteins orfragments identified as critical conserved effector domains.

FIG. 13A-C. Peptide antisera blocking reveals that the OmpA invasindomain lies within amino acids 59-74.

FIG. 14. Locations of linker insertion mutations that identify regionsrequired to disrupt the ability of OmpA to antagonize Aph infection,showing alignment of OmpA (SEQ ID NO: 10) with Anaplasma and Ehrlichiaspecies homologs AM854 (SEQ ID NO: 32), ACHIS_00486 (SEQ ID NO: 34),ECH_0462 (SEQ ID NO:40), Ecaj_0563 (SEQ ID NO:46), and Erum 5620 (SEQ IDNO:52) with regions of identity and similarity shaded.

FIG. 15. Percent of infection using linker insertion mutants of OmpA.

FIG. 16. Percent of infection in alanine substitution experiments thatidentified that OmpA aa59-74 are important for infection.

FIGS. 17A and B. ELISA results showing the specificity of antiserumraised against Asp14 aa98-112 or aa113-124.

FIG. 18. Percent of bacterial infection inhibited by pretreatment of Aphwith antiserum specific for Asp14 invasin domain.

FIG. 19. Percent of infection reduced by antisera specific for the OmpAinvasin domain, Asp14 invasin domain, or combinations thereof.

FIGS. 20A and B. Western blot and ELISA showing that A. phagocytophilumOmpA and A. marginale OmpA share B-cell epitopes.

DETAILED DESCRIPTION

Aspects of the invention are related to diagnosing, preventing, andtreating zoonotic diseases caused by Anaplasmataceae bacteria. Thediseases affect both animals and humans and are collectively referred toas anaplasmosis, but more specifically known as HGA when transmitted tohumans. Aph surface proteins OmpA and Asp14 have been identified asmediating bacteria-host cell binding and entry. Thus, the surfaceproteins OmpA and Asp14 and fragments thereof can be used for diagnosingwhether a patient has been suffering from Aph infection. Specifically,if antibodies to one or more of OmpA or Asp14 are identified in serum orother biological material from a subject suspected of an Aph infectionby suitable assay, such as ELISA or immunoblot, where, for example, theantibodies bind to or interact with OmpA or Asp14 proteins or fragmentsthereof, then it can be determined that the subject has been exposed to,infected with, or is currently infected with Aph. Furthermore,administration of OmpA or Asp14 proteins or fragments, or nucleic acidsencoding for OmpA or Asp14 proteins, such as in forms where the nucleicacids are present with a vector such as a viral vector, or are presentas purified peptides, polypeptides or proteins in a pharmaceuticallyacceptable carrier, can provide an immunogenic response in the subjectand protection from subsequent Aph infection, or provide for treatmentby the production of antibodies to Aph infection in a subject that isalready infected.

The critical regions of Asp14 and OmpA that mediate infection are highlyconserved among family members Aph, A. marginale, and closely relatedEhrlichia species, such as E. chaffeensis, E. canis, and E. ruminatium,and may be highly conserved in A. platys. In particular, Aph and A.marginale are closely related and express many gene homologs, includingAsp14, OmpA and other surface antigens. The high degree of conservationmakes these surface proteins ideal for producing a vaccine orimmunogenic composition to provide protection from or therapy formultiple pathogens in humans and animals.

In one embodiment, the composition of the invention comprises one ormore isolated and purified recombinant polypeptides. Each polypeptidecomprises amino acid sequences encoding an Asp14 or an OmpA invasindomain that mediates uptake of Aph bacteria into host cells. The Asp14invasin domain lies within aa113-124 (SEQ ID NO:03). In otherembodiments, polypeptide fragments such as Asp14 aa101-124 (SEQ IDNO:02) or the full length protein (SEQ ID NO:01) are used. In anotherembodiment, the composition of the invention comprises the invasindomain of OmpA, which lies within aa59-74 (SEQ ID NO:04). In otherembodiments, a larger fragment of OmpA encompassing aa19-74 (SEQ IDNO:05), or the full length OmpA protein (SEQ ID NO:06) is used. It iscontemplated that virtually any protein sequence, as well as itscorresponding nucleic acid sequence coding for the protein sequence thatis or includes SEQ ID NO: 04 may be used. This would include the fulllength sequence (e.g., SEQ ID NO:06) as well as any sequence of, forexample 5-50 (or less than 5 or more than 50) amino acids before thebeginning or at the end of the amino acid sequence defined by of SEQ IDNO:04, and this can include amino acids which are present in SEQ IDNO:06 as well as amino acids which are from different species (e.g., achimera) or from a synthetic sequence, e.g., a histidine or GST tag.While the invention may comprise one, or a plurality, or multiple copiesof any single one of these polypeptides, yet another embodiment is amixture of at least two of the polypeptides encoded by SEQ ID NO:01, 02,03, 04, 05 and 06. Any of these polypeptides may be produced by means ofchemical synthesis or recombinant techniques well-known to those ofordinary skill in the art of molecular biology.

There is currently no means for preventing transmission of the bacteriacausing anaplasmosis or HGA. While antibiotic treatments exist, thesetreatments are not advised for some groups of patients. In oneembodiment, the invention is a vaccine for prevention or treatment ofanaplasmosis and HGA. One embodiment of the invention is apharmaceutically acceptable composition comprising one or a plurality ofany one of or a mixture of at least two amino acid sequences which areor include the amino acid sequences which are identified as SEQ IDNO:01, 02, 03, 04, 05 and 06. Administration of the composition of theinvention stimulates an immune response in a subject and production ofantibodies against Asp14, OmpA, or both. Because Asp14 and OmpA are onthe outer surface of Aph bacteria, antibodies produced by the subjectwill block binding of bacteria to host cells and interfere with uptakeinto vacuoles. Bacteria unable to enter host cells will be detected bythe host immune system and cleared from the body. Blockade can occur atthe point of entry into neutrophils or endothelial cells or transferbetween these two host cell types. Interruption of the zoonotic lifecycle provides a further benefit to public health and well-being bybreaking the chain of disease transmission to others.

Aside from commercial assays to detect Apl in dogs, there is no specificassay to rapidly or confirm Anaplasmataceae infection, or accuratelydiagnose HGA or anaplasmosis. In another embodiment, the inventionprovides a method to detect the presence of Aph Asp14 or OmpA in assaysof biological samples obtained from subjects to bind to antibodiesproduced by an Anaplasmataceae-infected individual, either of whichwould be diagnostic for HGA or anaplasmosis. The preferred compositionfor diagnostic testing may comprise either full length Amp14 (SEQ ID NO:3) or OmpA (SEQ ID NO:06). However, compositions comprising fragments ofAmp14, such as SEQ ID NO:01 and/or 02, are also contemplated, as are anymixtures of at least two of SEQ ID NO:01, 02, and 03. Likewise,composition comprising fragments of OmpA, such as SEQ ID NO:04 and/or05, and any mixtures of at least two of SEQ ID NO:04, 05, and 06. Theassay used to detect antibodies may be any type of immunoassay, such asan immunoblot or an enzyme-linked immunosorbent assay. The test samplemay be any type of body fluid, such as blood, plasma, serum, urine,saliva, or other body fluid. Tissues or cells may also be used, such astissue sections or cell preparations adhered to slides or coverslips forimmunohistochemical staining. The preferred embodiment is an ELISA witheach protein type to independently detect antibodies to Asp14, and OmpA,however, a combination to detect Asp14 and OmpA antibodies in one ELISAis also contemplated.

In order to facilitate the understanding of the present invention, thefollowing definitions are provided:

Aph: Anaplasma phagocytophilum or A. phagocytophilum, an Anaplasmataseaefamily bacterium that is tick-born and causes anaplasmosis in humans andanimals.

Apl: Anaplasma platys or A. platys, an Anaplasmataseae family memberbacterium that is tick-born and causes anaplasmosis that is restrictedto dogs.

Anaplasmataceae: a family of closely related bacteria, includingAnaplasma and Ehrlichia species. The genera Neorickettsia and Wolbachhiaare also Anaplasmataceae, bacteria but do not cause anaplasmosis.

Antigen: term used historically to designate an entity that is bound byan antibody, and also to designate the entity that induces theproduction of the antibody. More current usage limits the meaning ofantigen to that entity bound by an antibody, while the word “immunogen”is used for the entity that induces antibody production. Where an entitydiscussed herein is both immunogenic and antigenic, reference to it aseither an immunogen or antigen will typically be made according to itsintended utility. The terms “antigen”, “antigenic region” “immunogen”and “epitope” may be used interchangeably herein. As used herein, anantigen, immunogen or epitope is generally a portion of a protein (e.g.a peptide or polypeptide).Asp14: 14-kilodalton Aph surface protein. OmpA homologs are expressed byAnaplasmataceae family members, including Aph, A. marginale, Ehrlichiachaffeensis, E. canis, E. ewingii, and E. ruminatium.OmpA: Outer membrane protein A. OmpA homologs are expressed byAnaplasmataceae family members, including Aph, A. marginale, Ehrlichiachaffeensis, E. canis, E. ewingii, and E. ruminatium.DC and RC: Aph undergoes a biphasic developmental cycle, the kinetics ofwhich have been tracked in promyelocytic HL-60 cells. The cycle beginswith attachment and entry of an infectious dense-cored (DC) organism.Once intracellular, the DC differentiates to the non-infectiousreticulate cell (RC) form and replicates by binary fission to produce abacteria-filled organelle called a morula. Later, the RCs transitionback to DCs, which initiate the next round of infection.Epitope: a specific chemical domain on an antigen that is recognized bya B-cell receptor, and which can be bound by secreted antibody. The termas used herein is interchangeable with “antigenic determinant”.Immunodominant epitope: The epitope on a molecule that induces thedominant, or most intense, immune response. The immunodominant epitopewould elicit the greatest antibody titer during infection orimmunization, as measured by, for example, the fraction of reactivityattributable to a certain antigen or epitope in an enzyme-linkedimmunosorbant assay as compared with the total responsiveness to anantigen set or entire protein.Invasin domain: An invasin domain is a region of a pathogen's proteinthat binds a host cell and mediates intracellular signaling and pathogenentry into the host cell. In some cases, uptake of the pathogen resultsin the formation of a vacuole in which the intracellular pathogen willreside. The invasin domains of the invention are linear amino acidsequences within Asp14, OmpA, or other surface proteins that are foundon the outer membrane of the bacteria Aph and other Anaplasmataceaefamily members, and can vary slightly from one family member to thenext. However, the invasin domain in each Asp14 homolog is critical foruptake of bacteria into host cells (known to be neutrophils andendothelial cells in the case of Anaplasmataceae).Linker sequences: short peptide sequences encoding functional units thatmay be engineered or otherwise added at the ends or within recombinantproteins, polypeptides, peptides of interest. Linker sequences may beused as “handles” for protein purification, as detectable signals ofexpression or binding to other proteins or macromolecules, to modulatetertiary structure, or enhance antigenicity. Examples of linkersequences include but are not limited to an amino acid spacer, an aminoacid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, and a protein purification ligand.LINKER: a program to generate linker sequences for fusion proteins.Protein Engineering 13(5): 309-312, which is a reference that describesunstructured linkers. Structured (e.g. helical) sequence linkers mayalso be designed using, for example, existing sequences that are knownto have that secondary structure, or using basic known biochemicalprinciples to design the linkers.Tags: Recombinant protein sequences that can be added to the N- orC-terminus of a recombinant protein for the purpose of identification orfor purifying the recombinant protein for subsequent uses. Examples ofrecombinant protein tags that may be useful in practicing the inventioninclude but are not limited to glutathione-S-transferease (GST),poly-histidine, maltose binding protein (MBP), FLAG, V5, halo, myc,hemaglutinin (HA), S-tag, calmodulin, tag, streptavidin binding protein(SBP), Softagl™, Softag3™, Xpress tag, isopeptag, Spy Tag, biotincarboxyl carrier protein (BCCP), GFP, Nus-tag, strep-tag, thioredoxintag, TC tag, and Ty tag. All such tags are well-known to those ofordinary skill in the art of recombinant protein production.Epitope: An epitope may comprise a single, non-interrupted, contiguouschain of amino acids joined together by peptide bonds to form a peptideor polypeptide. Such an epitope can be described by its primarystructure, i.e. the linear sequence of amino acids in the peptide chain.Epitope may also refer to conformational epitopes, which are comprisedof at least some amino acids that are not part of an uninterrupted,linear sequence of amino acids, but which are brought into proximity toother residues in the epitope by secondary, tertiary and/or quaternaryinteractions of the protein. Residues in conformational epitopes may belocated far from other resides in the epitope with respect to primarysequence, but may be spatially located near other residues in theconformational epitope due to protein folding.Protein: Generally means a linear sequence of about 100 or more aminoacids covalently joined by peptide bonds.Polypeptide: Generally means a linear sequence of about 55 to about 100amino acids covalently joined by peptide bonds.Peptide: Generally means a linear sequence of about 55 or fewer aminoacids covalently joined by peptide bonds.Note: The terms “peptide”, “polypeptide” and “protein” may be usedinterchangeably herein.Chimeric or fusion peptide or polypeptide: a recombinant or syntheticpeptide or polypeptide whose primary sequence comprises two or morelinear amino acid sequences which do not occur together in a singlemolecule in nature. The two or more sequences may be, for example, apeptide (e.g. an epitope or antigenic region) and a linker sequence, ortwo or more peptides (which may be the same or different) which areeither contiguous or separated by a linker sequences, etc.Tandem repeats: two or more copies of nucleic acid or amino acidsequences encoding the same peptide, which are arranged in a linearmolecule and are either contiguous or separated by a linker sequences,etc.Original or native or wild type sequence: The sequence of a peptide,polypeptide, protein or nucleic acid as found in nature.Recombinant peptide, polypeptide, protein or nucleic acid: peptide,polypeptide, protein or nucleic acid that has been produced and/ormanipulated using molecular biology techniques such as cloning,polymerase chain reaction (PCR), etc.Synthetic peptide, polypeptide, protein or nucleic acid: peptide,polypeptide, protein or nucleic acid that has been produced usingchemical synthesis procedures.Type-specific: associated primarily with a single phyletic group.Surface protein: A protein located on the outer surface membrane of acell or bacterium.

TABLE 1 Aph Sequence Listing with SEQ ID Numbers. GENBANK ACCES- SEQSSION ID PROTEIN # AND NO NAME NAME AMINO ACID SEQUENCE SEQ ID Full- YP_MIPLAPWKSISVVYMSGSDEYKEIIKQ NO: 01 length 504865CIGSVKEVFGEGRFDDVVASIMKMQE Asp14 APH_ KVLASSMQQDDTGTVGQIESGEGSGA 0248RLSDEQVQQLMNSIREEFKDDLRAIKR RILKLERAVYGANTPKES SEQ ID Asp14  APH_LRAIKRRILKLERAVYGANTPKES NO: 02 aa101- 0248 124 SEQ ID Asp 14  APH_RAVYGANTPKES NO: 03 aa113- 0248 124 SEQ ID Full  YP_MLRRSSFFCLLALLSVTSCGTLLPDSN NO: 04 length 504946VGVGRHDLGSHRSVAFAKKVEKVYF OmpA APH_ DIGKYDLKGPGKKVILELVEQLRQDD 0338SMYLVVIGHADATGTEEYSLALGEKR ANAVKQFIIGCDKSLAPRVTTQSRGKAEPEVLVYSTDAQEVEKANAQNRRA VIVVEFAHIPRSGVADMHAPVASSITSENSNASAEGEDMEASEFSSAIAN SEQ ID OmpA  APH_ CGTLLPDSNVGVGRHDLGSHRSVAFANO: 05 aa19- 0338 KKVEKVYFDIGKYDLKGPGKKVILEL 74 VEQLR SEQ ID OmpA  APH_LKGPGKKVILELVEQL NO: 06 aa59- 0338 74 SEQ ID OmpA  APH_ EKVYFDIGK NO: 07aa48- 0338 56 SEQ ID OmpA APH_ GHADATGTEEYSLALG NO: 08 0338 SEQ ID OmpAAPH_ LVYSTDAQEVEKANAQNRRAV NO: 09 0338 SEQ ID OmpA APH_PDSNVGVGRHDLGSHRSVAFAKKVE NO: 10 0338 KVYFDIGKYDLKGPGKKVILELVEQLRQDDSMYLVVIGHADATGTEEYSLAL GEKRANAVKQFHGCDKSLAPRVTTQSRGKAEPEVLVYSTDAQEVEKANAQN RRAVIVVE FAHIPRSGVADM SEQ ID Asp14  APH_LRAIKRRILKLE NO: 11 aa101- 0248 112 SEQ ID Asp14  APH_DEYKEIIKQCIGSVKEVFGEGRFDDVV NO: 12 aa19- 0248 ASIMKMQEKVLASSM 60

TABLE 2 Asp14 Homologs Sequence Listing with SEQ  ID Numbers SEQ IDAnaplasma AM936 MSGEDEYKEIIRQCIGSVKEVFG NO: 13 marginaleEGRFDDVVASIMKMQEKVLASSM KDGDPVGQIAADGVGNELYDRIA DRLEERVSQKISEDLRIIKKRLLRLERVVLGGGSVSGDAAAHQVSG NQPSQQNSSAAAEGG SEQ ID A.  AM936LGGGSVSGDAAAHQVSGNQPSQQ NO: 14 marginale NSSAAAEGG SEQ ID A.  ACIS_MSGEDEYKEIIRQCIGSVKEVFG NO: 15 marginale 00403 EGRFDDVVASIMKMQEKVLASSMsubspecies KDGDPVGQIAADGVGNELYDRIA Centrale DRLEERVSQKISEDLRIIKKRLLRLERVVLGGGSVSGDAAAAHQVS GNQPSQQNSSAAAEGG SEQ ID A.  ACIS_LGGGSVSGDAAAAHQVSGNQPSQ NO: 16 marginale 00403 QNSSAAAEGG subspeciesCentrale SEQ ID A.  AM936 & MSGEDEYKEIIRQCIGSVKEVFG NO: 17 marginale &ACIS- EGRFDDVVASIMKMQEKVLASSM A.  00403 marginale subspecies CentraleSEQ ID A.  AM936 & DLRIIKKRLLRLERVV NO: 18 marginale & ACIS- A.  00403marginale subspecies Centrale SEQ ID Ehrlichia ECH_MAEDDYKGVIKQYIDTVKEIVGD NO: 19 chaffeensis 0377 SKTFDQMFESVVRIQERVMAANAQNNEDGVIDNGDQVKRIGSSTSE SISNTEYKELMEELKVIKKRILR LERKILKPKEEV SEQ ID E. ECH_ MAEDDYKGVIKQYIDTVKEIVGD NO: 20 chaffeensis 0377 SKTFDQMFESVVRIQERVMSEQ ID E.  ECH_ ELKVIKKRILRLE NO: 21 chaffeensis 0377 SEQ ID E.  ECH_RKILKPKEEV NO: 22 chaffeensis 0377 SEQ ID E. canis Ecaj_MADDEYKGVIQQYINTVKEIVSD NO: 23 0636 SKTFDQMFESVVKIQERVMEANAQNDDGSQVKRIGSSTSDSISDSQ YKELIEELKVIKKRLLRLEHKVL KPKEGA SEQ ID E. canisEcaj_ MADDEYKGVIQQYINTVKEIVSD NO: 24 0636 SKTFDQMFESVVKIQERVM SEQ IDE. canis Ecaj_ ELKVIKKRLLRLE NO: 25 0636 SEQ ID E. canis Ecaj_HKVLKPKEGA NO: 26 0636 SEQ ID E.  Erum MADEDYKGVIKQYIDTVKEIVGD NO: 27ruminantium 6320 SKTFDQMFESVVKIQERVMAASA QNEANGALVEGDSKMKRIRSADDSIAYTQSQELLEELKVLKKRIAR LERHVFKSNKTEA SEQ ID E.  ErumMADEDYKGVIKQYIDTVKEIVGD NO: 28 ruminantium 6320 SKTFDQMFESVVKIQERVMSEQ ID E.  Erum ELKVLKKRIARLE NO: 29 ruminantium 6320 SEQ ID E.  ErumRHVFKSNKTEA NO: 30 ruminantium 6320

TABLE 3 OmpA Homologs Sequence Listing with SEQ  ID Numbers SEQ IDAnaplasma AM854 MLHRWLALCFLASFAVTGCGLFS NO: 31 marginaleKEKVGMDIVGVPFSAGRVEKVYF DFNKYEIKGSGKKVLLGLVERMK ADKRSTLLIIGHTDSRGTEEYNLALGERRANAVKEFILGCDRSLSP RISTQSRGKAEPEVLVYSSDFKE AEKAHAQNRRVVLIVECQHSVSPKKKMAIKWPFSFGRSAAKQDDGV SSEVSDENPVDDSSEGIASEEAA PEEGVVSEEAAEEAPEVAQDSSAGVVAPE SEQ ID A.  AM854 LFSKEKVGMDIVGVPFSAGRVEK NO: 32 marginaleVYFDFNKYEIKGSGKKVLLGLVE RMKADKRSTLLII SEQ ID A.  ACIS_MLHRWLALCLLASLAVTGCELFN NO: 33 marginale 00486 KEKVNIDIGGVPLSAGRVEKVYFsubspecies DFNKYEIKGSGKKVLLGLVERMK Centrale ADKMSTLLIVGHTDSRGTEEYNLALGERRANAVKEFILGCDRSLSP RISTQSRGKAEPEILVYSSDFKE AHAQNRAEKRVVLIMECQHAASPKKARVSRWPFSFGRSSATQQDNG GGTVAAGSPGEDAPAEVVEPEET QEAGE SEQ ID A.  ACIS_LFNKEKVNIDIGGVPLSAGRVEK NO: 34 marginale 00486 VYFDFNKYEIKGSGKKVLLGLVEsubspecies RMKADKMSTLLIV Centrale SEQ ID A.  AM854 &AGRVEKVYFDFNKYEIKGSGKKV NO: 35 marginale &  ACIS- LLGLVERMKAD A. 00486marginale subspecies Centrale SEQ ID A.  AM936 & GHTDSRGTEEYNLALG NO: 36marginale &  ACIS- A. 00403 marginale subspecies Centrale SEQ ID A. AM854 & RRANAVKEFILGCDRSLSPRIST NO: 37 marginale &  ACIS- QSRGKAE A.00486 marginale subspecies Centrale SEQ ID A.  AM854 &LVYSSDFKEAEKAHAQNRRVVLI NO: 38 marginale &  ACIS- A. 00486 marginalesubspecies Centrale SEQ ID Ehrlichia ECH_ MKHKLVFIKFMLLCLILSSCKTT NO: 39chaffeensis 0462 DHVPLVNVDHVFSNTKTIEKIYF GFGKATIEDSDKTILEKVMQKAEEYPDTNIIIVGHTDTRGTDEYNL ELGKQRANAVKDFILERNKSLED RIIIESKGKSEPAVLVYSNNPEEAEYAHTKNRRVVITLTDNLIYKA KSSDKDPSSNKTEQ SEQ ID Ehrlichia ECH_NVDHVFSNTKTIEKIYFGFGKAT NO: 40 chaffeensis 0462 IEDSDKTILEKVMQKAEEYPDTNIIIV SEQ ID Ehrlichia ECH_ IEDSDKTILEKVMQKAEEYPDTN NO: 41 chaffeensis0462 IIIV SEQ ID Ehrlichia ECH_ GHTDTRGTDEYNLELGE NO: 42 chaffeensis0462 SEQ ID Ehrlichia ECH_ QRANAVKDFILERNKSLEDRIII NO: 43 chaffeensis0462 ESKGKSEPAV SEQ ID Ehrlichia ECH_ LVYSNNPEEAEYAHTKNRRVVI NO: 44chaffeensis 0462 SEQ ID E. canis Ecaj_ MKHKLVFIKFILLCLILSSCKTT NO: 450563 DHVPLVNTDHVFSNMKTIEKIYF DFGKATIGDSDKAILEKVIQKAQKDTNTNIVIVGHTDTRGTDEYNL ELGEQRANAVKDFIIEHDKSLEN RITVQSKGKSEPAVLVYSSNPEEAEHAHAKNRRVVITLTDNGNKTS Q SEQ ID E. canis Ecaj_ TTDHVPLVNTDHVFSNMKTIEKINO: 46 0563 YFDFGKATIGDSDKAILEKVIQK AQKDTNTNIVIV SEQ ID E. canis Ecaj_GDSDKAILEKVIQKAQKDTNTNI NO: 47 0563 VIV SEQ ID E. canis Ecaj_GHTDTRGTDEYNLELGE NO: 48 0563 SEQ ID E. canis Ecaj_QRANAVKDFIIEHDKSLENRITV NO: 49 0563 QSKGKSEPAV SEQ ID E. canis Ecaj_LVYSSNPEEAEHAHAKNRRVVI NO: 50 0563 SEQ ID E.  ErumMRYQLIVANLILLCLTLNGCHFN NO: 51 ruminantium 5620 SKHVPLVNVHNLFSNIKAIDKVYFDLDKTVIKDSDKVLLEKLVQKA QEDPTTDIIIVGHTDTRGTDEYN LALGEQRANAVRDFIISCDKSLEKRITVRSKGKSEPAILVYSNNPK EAEDAHAKNRRVVITLVNNSTST DNKVPTTTTPFNEEAHNTISKDQENNTQQQAKSDNINNINTQQKLE QDNNNTPEVN SEQ ID E.  ErumNSKHVPLVNVHNLFSNIKAIDKV NO: 52 ruminantium 5620 KYFDLDTVIKDSDKVLLEKLVQKAQEDPTTDIIIV SEQ ID E.  Erum DSDKVLLEKLVQKAQEDPTTDII NO: 53 ruminantium5620 IV SEQ ID E.  Erum GHTDTRGTDEYNLALGE NO: 54 ruminantium 5620 SEQ IDE.  Erum QRANAVRDFIISCDKSLEKRITV NO: 55 ruminantium 5620 RSKGKSEPAISEQ ID E.  Erum LVYSNNPKEAEDAHAKNRRVVI NO: 56 ruminantium 5620In addition to sequences for OmpA and Asp14 shown in Table 1, andhomologs shown in Tables 2-3, other surface proteins that Aphpreferentially expresses in human versus tick cells may be used. Table 4shows examples of proteins that can be included in the “cocktail” ofpeptides, polypeptides or protein sequences of the composition of theinvention. Examples of these include APH_0915, APH_1325 (Msp2),APH_1378, APH_1412, APH_0346, APH_0838, APH_0839, APH_0874, and APH_0906because all are upregulated 3- to 60-fold during RC-DC transition, DCexit, and/or reinfection and our surface proteomic study indicates thatthey are surface proteins. The file names for each of the aforementionedproteins are from the A. phagocytophilum HZ annotated genome. A similarexpression profile is exhibited by APH_1235, which is another late stagegene that is upregulated 70-fold, as taught by Mastronunzio andcolleagues, who identified APH_1235 as an A. phagocytophilum surfaceprotein. P44 is a 44 kilodalton surface protein and is the bacterium'smajor surface protein. Synonyms of P44 are Msp2 (major surface protein2) and Msp2 (P44). All Anaplasma species encode P44 proteins and thereare huge repertoires of P44 genes in these bacterial species'chromosomes. For instance, the annotated Aph strain HZ genome encode 113P44 proteins. These exist as complete genes or pseudogenes (incompletegenes). There is one expression site for p44 genes. Basically, differentp44 genes get shuffled into the expression site by a process known asgene conversion with the end result being that Aph (and other Anaplasmaspecies) can vary the P44 protein on their cell surfaces, a processcalled antigenic variation. This enables them to perpetually evade thehumoral immune response.

TABLE 4 Anaplamatacaea Surface Proteins Sequence Listing and SEQ IDNumbers SEQ ID NO: 57 Full-length APH_0915 Genbank Accession No:YP_505488 SEQ ID NO: 58 Full-length APH_1378 Genbank Accession No:YP_505877 SEQ ID NO: 59 Full-length APH_1412 Genbank Accession No:YP_505903 SEQ ID NO: 60 Full-length APH_0346 Genbank Accession No:YP_504953 SEQ ID NO: 61 Full-length APH_0838 Genbank Accession No:YP_505415 SEQ ID NO: 62 Full-length APH_3839 Genbank Accession No:YP_505416 SEQ ID NO: 63 Full-length APH_0874 Genbank Accession No:YP_505450 SEQ ID NO: 64 Full-length APH_0906 Genbank Accession No:YP_505479 SEQ ID NO: 65 Full-length APH_1325 Genbank Accession No:(Msp2) YP_505833 SEQ ID NO: 66 Full-length APH_1235 Genbank AccessionNo: YP_505764In addition to polypeptides sequences from Aph surface proteins, othersequences may be included in the polypeptides of the invention. Suchsequences include but are not limited to antigenic peptide sequencessuch as linker sequences which in and of themselves are antigenic.Examples of recombinant protein tags that may be useful in practicingthe invention include but are not limited to glutathione-S-transferease(GST), poly-histidine, maltose binding protein (MBP), FLAG, V5, halo,myc, hemaglutinin (HA), S-tag, calmodulin, tag, streptavidin bindingprotein (SBP), Softagl™, Softag3™, Xpress tag, isopeptag, Spy Tag,biotin carboxyl carrier protein (BCCP), GFP, Nus-tag, strep-tag,thioredoxin tag, TC tag, and Ty tag. Examples of linker sequencesinclude but are not limited to an amino acid spacer, an amino acidlinker, a signal sequence, a stop transfer sequence, a transmembranedomain, and a protein purification ligand. It should also be recognizedthat a multitude of other such sequences are known to those of skill inthe art, and inclusion of other antigenic, linker, or tag sequences iscontemplated.

Those of skill in the art will recognize that, while in some embodimentsof the invention, the amino acid sequences that are chosen for inclusionin the polypeptides of the invention correspond exactly to the primaryamino acid sequence of the original or native sequences of an Asp14 orOmpA protein, this need not always be the case. The amino acid sequenceof an epitope that is included in the polypeptides of the invention maybe altered somewhat and still be suitable for use in the presentinvention. For example, certain conservative amino acid substitutionsmay be made without having a deleterious effect on the ability of thepolypeptides to elicit an immune response. Those of skill in the artwill recognize the nature of such conservative substitutions, forexample, substitution of a positively charged amino acid for anotherpositively charged amino acid (e.g. K for R or vice versa); substitutionof a negatively charged amino acid for another negatively charged aminoacid (e.g. D for E or vice versa); substitution of a hydrophobic aminoacid for another hydrophobic amino acid (e.g. substitution of A, V, L,I, W, etc. for one another); etc. All such substitutions or alterationsof the sequences of the polypeptides that are disclosed herein areintended to be encompassed by the present invention, so long as theresulting polypeptides still function to elicit a suitable immuneresponse. In addition, the amino acid sequences that are included in thepolypeptides or any chimeric proteins of the invention need notencompass a full length native polypeptide. Those of skill in the artwill recognize that truncated versions of amino acid sequences that areknown to be or to contain antigenic polypeptides may, for a variety ofreasons, be preferable for use in the practice of the invention, so longas the criteria set forth for an epitope is fulfilled by the sequence.Amino acid sequences that are so substituted or otherwise altered may bereferred to herein as “based on” or “derived from” the original wildtype or native sequence. In general, the Asp14 or OmpA proteins orpolypeptide fragments from which the linear epitopes are “derived” or onwhich the linear epitopes are “based” are the Asp14 or OmpA proteins orpeptide fragments as they occur in nature. These natural Asp14/OmpAproteins may alternatively be referred to as native or wild typeproteins.

Such changes to the primary sequence may be introduced for any of avariety of reasons, for example, to eliminate or introduce a proteasecleavage site, to increase or decrease solubility, to promote ordiscourage intra- or inter-molecular interactions such as folding, ionicinteractions, salt bridges, etc, which might otherwise interfere withthe presentation and accessibility of the individual epitopes along thelength of a peptide or polypeptide. All such changes are intended to beencompassed by the present invention, so long as the resulting aminoacid sequence functions to elicit a protective antibody response in ahost to whom it is administered. In general, such substituted sequenceswill be at least about 50% identical to the corresponding sequence inthe native protein, preferably about 60 to 70, or even 70 to 80, or 80to 90% identical to the wild type sequence, and preferably about 95, 96,97, 98, 99, or even 100% identical to a native Asp14 or OmpA sequence orpeptide fragment. The reference native Asp14 or OmpA sequence or peptidefragment may be from any suitable type of Anaplasmataceae, e.g. from anyAnaplasmataceae which is known to infect mammals.

In some embodiments of the invention, individual linear epitopes in achimeric vaccinogen are separated from one another by interveningsequences that are more or less neutral in character, i.e. they do notin and of themselves elicit an immune response to Anaplasmataceae. Suchsequences may or may not be present between the epitopes of a chimera.If present, they may, for example, serve to separate the epitopes andcontribute to the steric isolation of the epitopes from each other.Alternatively, such sequences may be simply artifacts of recombinantprocessing procedures, e.g. cloning procedures. Such sequences aretypically known as linker or spacer peptides, many examples of which areknown to those of skill in the art. See, for example, Crasto, C. J. andJ. A. Feng. 2000.

In addition, other elements may be present in chimeric proteins, forexample leader sequences or sequences that “tag” the protein tofacilitate purification or detection of the protein, examples of whichinclude but are not limited to tags that facilitate detection orpurification (e.g. S-tag, or Flag-tag), other antigenic amino acidsequences such as known T-cell epitope containing sequences and proteinstabilizing motifs, etc. In addition, the chimeric proteins may bechemically modified, e.g. by amidation, sulfonylation, lipidation, orother techniques that are known to those of skill in the art.

The invention further provides nucleic acid sequences that encodechimeric proteins of the invention. Such nucleic acids include DNA, RNA,and hybrids thereof, and the like. Further, the invention comprehendsvectors which contain or house such coding sequences. Examples ofsuitable vectors include but are not limited to plasmids, cosmids, viralbased vectors, expression vectors, etc. In a preferred embodiment, thevector will be a plasmid expression vector.

The chimeric proteins of the invention may be produced by any suitablemethod, many of which are known to those of skill in the art. Forexample, they may be chemically synthesized, or produced usingrecombinant DNA technology (e.g. in bacterial cells, in cell culture(mammalian, yeast or insect cells), in plants or plant cells, or bycell-free prokaryotic or eukaryotic-based expression systems, by otherin vitro systems, etc.). In some embodiments, the polypeptides areproduced using chemical synthesis methods.

The present invention also provides compositions for use in eliciting animmune response. The compositions may be utilized as vaccines to preventor treat anaplasmosis, particularly when manifested in humans as HGA. Byeliciting an immune response, we mean that administration of the antigencauses the synthesis of specific antibodies (at a titer as describedabove) and/or cellular proliferation, as measured, e.g. by ³H thymidineincorporation, or by other known techniques. By “vaccine” we mean alinear polypeptide, a mixture of linear polypeptides or a chimeric orfusion polypeptide that elicits an immune response, which results inprotection of an organism against challenge with an Anaplasmataceaespecies bacterium. The protective response either wholly or partiallyprevents or arrests the development of symptoms related to anaplasmosisor HGA infection (i.e. the symptoms of anaplasmosis), in comparison to anon-vaccinated (e.g. adjunct alone) control organisms, in which diseaseprogression is not prevented. The compositions include one or moreisolated and substantially purified polypeptides or chimeric peptides asdescribed herein, and a pharmacologically suitable carrier. Thepolypeptides or chimeric peptides in the composition may be the same ordifferent, i.e. the composition may be a “cocktail” of differentpolypeptides or chimeric peptides, or a composition containing only asingle type of polypeptide or chimeric peptide. The preparation of suchcompositions for use as vaccines is well known to those of skill in theart. Typically, such compositions are prepared either as liquidsolutions or suspensions, however solid forms such as tablets, pills,powders and the like are also contemplated. Solid forms suitable forsolution in, or suspension in, liquids prior to administration may alsobe prepared. The preparation may also be emulsified. The activeingredients may be mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredients. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanoland the like, or combinations thereof. In addition, the composition maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents, and the like. The vaccinepreparations of the present invention may further comprise an adjuvant,suitable examples of which include but are not limited to Seppic, QuilA, Alhydrogel, etc. If it is desired to administer an oral form of thecomposition, various thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders and the like may be added. The composition ofthe present invention may contain any such additional ingredients so asto provide the composition in a form suitable for administration. Thefinal amount of polypeptides or chimeric peptides in the formulationsmay vary. However, in general, the amount in the formulations will befrom about 0.01-99%, weight/volume.

The methods involve administering a composition comprising recombinantpolypeptides or chimeric peptides in a pharmacologically acceptablecarrier to a mammal. The mammal may be a human, but this need not alwaysbe the case. Because anaplasmosis is a zoonotic disease that causesanaplasmosis in all known mammalian hosts, veterinary applications ofthis technology are also contemplated. The vaccine preparations of thepresent invention may be administered by any of the many suitable meanswhich are well known to those of skill in the art, including but notlimited to by injection, inhalation, orally, intranasally, by ingestionof a food product containing the polypeptides or chimeric peptides, etc.In some embodiments, the mode of administration is subcutaneous orintramuscular. In addition, the compositions may be administered inconjunction with other treatment modalities such as substances thatboost the immune system, various anti-bacterial chemotherapeutic agents,antibiotics, and the like.

The present invention provides methods to elicit an immune response toAnaplasmataceae and/or to vaccinate against Anaplasmataceae infection inmammals. In one embodiment, the mammal is a human. However, those ofskill in the art will recognize that other mammals exist for which suchvaccinations would also be desirable, e.g. the preparations may also beused for veterinary purposes. Examples include but are not limited tocompanion “pets” such as dogs, cats, etc.; food source, work andrecreational animals such as cattle, horses, oxen, sheep, pigs, goats,and the like; or even wild animals that serve as a reservoir ofAnaplasmataceae, particularly wild animals adapted to living in closeproximity to urban areas (e.g. mice, deer, rats, raccoons, opossum,coyotes, etc).

The invention also provides a diagnostic and a method for using thediagnostic to identify individuals who have antibodies to the epitopescontained within the polypeptides or chimeric proteins of the invention.A biological sample from an individual (e.g. a human, a deer, or othermammals susceptible to infection by Anaplasmataceae) suspected of havingbeen exposed to Anaplasmataceae, or at risk for being exposed toAnaplasmataceae, is contacted with the peptides, polypeptides, orchimeric proteins of the invention. Using known methodology, thepresence or absence of a binding reaction between the polypeptides orchimeric proteins and antibodies in the biological sample is detected. Apositive result (i.e. binding occurs, thus antibodies are present)indicates that the individual has been exposed to and/or is infectedwith Anaplasmataceae. Further, the diagnostic aspects of the inventionare not confined to clinical use or home use, but may also be valuablefor use in the laboratory as a research tool, e.g. to identifyAnaplasmataceae bacteria isolated from ticks, to investigate thegeographical distribution of Anaplasmataceae species and strains, etc.

The present invention also encompasses antibodies to the epitopes and/orto the polypeptides or chimeric proteins disclosed herein. Suchantibodies may be polyclonal, monoclonal or chimeric, and may begenerated in any manner known to those of skill in the art. In apreferred embodiment of the invention, the antibodies are bactericidal,i.e. exposure of Anaplasmataceae bacteria to the antibodies causes deathof the bacteria. Such antibodies may be used in a variety of ways, e.g.as detection reagents to diagnose prior exposure to Anaplasmataceae, asa reagent in a kit for the investigation of Anaplasmataceae, to treatAnaplasmataceae infections, etc.

Alternatively, appropriate antigen fragments or antigenic sequences orepitopes may be identified by their ability, when included inpolypeptides or chimeric proteins, to elicit suitable antibodyproduction to the epitope in a host to which the polypeptides orchimeric proteins are administered. Those of skill in the art willrecognize that definitions of antibody titer may vary. Herein, “titer”is taken to be the inverse dilution of antiserum that will bind one halfof the available binding sites on an ELISA well coated with 100 ng oftest protein. In general, suitable antibody production is characterizedby an antibody titer in the range of from about 100 to about 100,000,and preferably in the range of from about 10,000 to about 10,000,000.Alternatively, and particularly in diagnostic assays, the “titer” shouldbe about three times the background level of binding. For example, to beconsidered “positive”, reactivity in a test should be at least threetimes greater than reactivity detected in serum from uninfectedindividuals. Preferably, the antibody response is protective, i.e.prevents or lessens the development of symptoms of disease in avaccinated host that is later exposed to Anaplasmataceae, compared to anunvaccinated host.

The following Examples are provided to illustrate various embodiments ofthe invention, however, as described in detail above, aspects of theinvention can be practiced in a variety of ways different from thoseillustrated in the Examples.

EXAMPLES

The following experimental procedures were used in the examples of theinvention:

Cell Lines and Cultivation of Uninfected and Aph-Infected HL-60 Cells.

PSGL-1 CHO cells and RF/6A cells were cultivated as described [21,77].Uninfected HL-60 cells (American Type Culture Collection [ATCC];Manassas, Va.; ATCC code CCL-240) and HL-60 cells infected with the AphNCH-1 strain or a transgenic HGE1 strain expressing GFP (a gift fromUlrike Munderloh of the University of Minnesota, Minneapolis, Minn.)were cultivated. Spectinomycin (Sigma-aldrich, St. Louis, Mo.) was addedto HL-60 cultures harboring transgenic HGE1 bacteria at a finalconcentration of 100 μg/ml.

Aph DC Organism Surface Biotinylation and Affinity Purification.

Aph DC organisms from 10⁹ infected (≥90%) HL-60 cells were enriched forby sonication followed by differential centrifugation as described [61].To purify DC organisms away from the majority of contaminating host andRC organism cellular debris, the sonicate was fractionated usingdiscontinuous Renografin (diatrizoate sodium, Bracco diagnostics,Princeton, N.J.) density gradient centrifugation. Purified DC organismswere resuspended in 1 ml of phosphate-buffered saline (PBS) (pH 8.0)containing 1 mM MgCl₂ and 10 mM Sulfo-NHS-SS-Biotin (Pierce; Rockland,Ill.) and incubated for 30 min at room temperature. Free biotin wasquenched by washing the sample with 50 mM Tris (pH 8.0), followed by twowashes with PBS. Biotinylated bacteria were solubilized inradioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl [pH 7.6],150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecylsulfate [SDS], 1 mM sodium orthovanadate, 1 mM sodium fluoride, andComplete EDTA-free protease inhibitor set cocktail [Roche, Indianapolis,Ind.]) on ice for 1 h. Every 20 min during the 1-h incubation, thesample was subjected to eight 8-s bursts on ice interspersed with 8-srest periods using a Misonix 54000 ultrasonic processor (Farmingdale,N.Y.) on an amplitude setting of 30. Insoluble material was removed byspinning at 10,000×g for 10 min at 4° C. To purify biotinylatedproteins, the clarified lysate was mixed with High Capacity NeutrAvidinagarose beads (Pierce) by end-over-end rotation overnight at 4° C. Thegel slurry was pelleted by centrifugation at 1,000×g for 1 min. Afterremoval of the supernatant, the beads were resuspended in eight ml PBSand parceled into ten 800 μl aliquots, each of which were added to spincolumns optimized for affinity purification (Pierce). The columns werewashed three times with PBS and centrifuged at 1,000×g to remove anynon-biotinylated proteins. The captured biotinylated proteins wereeluted from the beads by end-over-end rotation with 150 mM DTT in 0.25%sodium deoxycholate for 2 h at room temperature. The agarose beads werecentrifuged at 1,000×g for 2 min and the supernatant containing thebiotinylated proteins was saved. The Bradford assay was used todetermine the protein concentration of the eluate. The majority of thesample was stored at 4° C. until analysis. To ensure that this procedurehad enriched for DC bacterial surface proteins, an aliquot of theaffinity-purified sample was resolved by SDS-PAGE alongside an Aphwhole-cell lysate, neutravidin beads plus unlabeled DC whole celllysate, and neutravidin beads alone followed by silver staining.

2D-LC/MS-MS Proteome Analysis.

Unless otherwise stated, all buffers were made with LC/MS grade solvents(Fisher Chemical, Fairlawn, N.J.). Samples were processed for proteomicanalysis as described previously with the following methodologicaldetails. Following biotinylation enrichment of Aph surface proteins, 300μg of protein mass in 400 μl of lysis buffer was concentrated andexchanged into 25 μl of ammonium bicarbonate buffer (ABC) (50 mMNH₄CO₃/0.05% C₂₄H₃₉O₄Na) using a Centriprep YM-10 filter unit(Millipore, Billerica, Mass.). DTT was added to achieve a finalconcentration of 20 mM, and disulfide bonds were reduced at 90° C. for30 min. After cooling to room temperature, cysteine alkylation wasperformed on the sample with freshly prepared iodoacetamide (32 mM) for30 min at room temperature in the dark. Trypsin Gold (100 ng/μl;Promega, Madison, Wis.) was added to a final 1:100 enzyme:protein ratio,and the sample was incubated at 37° C. overnight. The digested samplewas dried within a speed vacuum and stored dry at −20° C.

The digest sample was reconstituted in 60 μL of 100 mM ammonium formate(pH 10) for multidimensional peptide separation and mass spectrometryanalysis on a 2D-nanoAcquity chromatography system online with a Synaptquadrupole/time-of-flight tandem mass spectrometer (Waters) aspreviously reported. Two-replicate injections were analyzed for thesample. Resulting data were processed using PLGS software, v2.4 (Waters)as described elsewhere. Data were then search against an Aph-specificFASTA database (RefSeq and Uniprot sources; downloaded February 2010)and its reversed-sequences as a decoy database. Search parametersrequired a minimum precursor ion intensity of 500 counts, two or morepeptide sequences per protein and a minimum of seven matching fragmentions. Trypsin selectivity was specified allowing for 1 missed cleavageevent and variable methionine oxidation. Using a decoy-database method,a score threshold was calculated at the 5% false-discovery rate.Confidence in the protein identification is also increased for thosethat were identified against both RefSeq and Uniprot Aph databases.

Analyses of Differential Aph Gene Expression Over the Course ofInfection.

Synchronous infections of HL-60 cells with Aph DC organisms wereestablished. Indirect immunofluorescence microscopic examination ofaliquots recovered at 24 h confirmed that ≥60% of HL-60 cells containedmorulae and that the mean number of morulae per cell was 2.8±0.6. Theinfection time course proceeded for 36 h at 37° C. in a humidifiedatmosphere of 5% CO₂. At the appropriate time-point, aliquots wereremoved and processed for RNA isolation and RT-qPCR was performed usinggene-specific primers. Relative transcript levels for each target werenormalized to the transcript levels of the Aph 16S rRNA gene (Aph_1000)using the 2^(−ΔC) _(T) method.

Transmission feeding of Aph infected Ixodes. scapularis nymphs.Aph-infected I. scapularis nymphs were obtained from a tick colonymaintained at Yale University (New Haven, Conn.). To propagateAph-infected ticks, clean I. scapularis larvae were fed on Aph-infectedC3H/HeJ mice, and the larvae were allowed to molt to nymphs. Infectionwas confirmed by testing 10% of each tick batch by PCR of the Aph 16SrRNA gene. Ticks were incubated at 23° C. with 85% relative humiditybetween feedings. To collect transmission-fed nymphs, groups of 20-25infected tick nymphs were placed to feed on clean 5-6 week-old C3H/HeJfemale mice and removed after 24, 48, or 72 hours of feeding. Salivaryglands dissected from 2-3 ticks were pooled into a tube of RLT bufferand frozen at −80° C., prior to RNA extraction with the Qiagen RNEasyKit (Qiagen, CA). Unfed ticks were dissected and RNA extracted fromcombined salivary glands and midguts. RT-qPCR was performed as describedabove.

Recombinant Protein Expression and Purification and Antisera Production.

Aph genes of interest were amplified using gene-specific primers andPlatinum Pfx DNA polymerase (Invitrogen). Amplicons were cloned intopENTR/TEV/D-TOPO (Invitrogen) as described [83] to yield pENTR-candidategene entry plasmids containing the genes of interest. Plasmid insertswere verified and recombination of the candidate gene insert downstreamof and in frame with the gene encoding GST was achieved using thepDest-15 vector (Invitrogen). In some cases plasmids encoding GST-OmpAor GST-Asp14 were subjected to PCR mutagenesis using the StratageneQuick Change kit according to the manufacturer's instructions for thepurpose of inserting DNA segments encoding five-amino acid linkers orsubstituting the alanine codon for a specific OmpA or Asp14 amino acid.Expression and purification of GST-OmpA, GST-Asp14, and GST-Msp5 andgeneration of murine polyclonal antisera against each protein wereperformed as described. KLH-conjugated peptides corresponding to OmpAamino acids 23-40, 41-58, or 59-74 or Asp14 amino acids 101-112 or113-124 were synthesized by and used for raising rabbit polyclonalantiserum against each peptide by New England Peptides (Gardner, Mass.).

Antibodies, Western Blot Analyses, and Spinning Disk ConfocalMicroscopy.

Antisera generated in this study and previous studies targeted OmpA,Asp14, Msp5, APH_0032 [61], APH_1387 [83], Msp2 (P44), and Asp55 andAsp62. The latter two antibodies were gifts from Yasuko Rikihisa of TheOhio State University (Columbus, Ohio). Anti-Msp2 (P44) mAb 20B4 [84,85]was a gift from J. Stephen Dumler of The Johns Hopkins University(Baltimore, Md.). Western blot analyses were performed. Aph infectedHL-60 cells were processed and analyzed via indirect immunofluorescenceusing spinning disk confocal microscopy.

Surface Trypsin Digestion of Intact Aph DC Organisms.

Intact DC bacteria were incubated at a 10:1 ratio of total protein totrypsin (Thermo Scientific, Waltham, Mass.) in IX PBS or vehicle aloneat 37° C. After 30 min, phenylmethanesulfonyl fluoride (Sigma) was addedto a final concentration of 2 mM. Bacteria were pelleted at 5,000 g for10 min, after which pellets were resuspended in urea lysis buffer andprocessed. Lysates of trypsin- and vehicle-treated Aph organisms werefractionated by SDS-PAGE, Western-blotted, and screened with antibodiestargeting OmpA, Asp14, Asp55 [33], Msp5, Msp2 (P44), and APH_0032.

Flow Cytometry.

1×10⁷ HL-60 cells infected with either transgenic HGE1 organismsexpressing GFP or wild-type Aph bacteria were mechanically lysedfollowed by differential centrifugation to pellet host cellular debris.GFP-positive Aph organisms and remaining host cellular debris werepelleted, followed by resuspension in PBS containing equivalent amountsof a 1:25 dilution of preimmune mouse serum, mouse anti-Asp14 oranti-OmpA, or secondary antibody alone. Antibody incubations and washsteps were performed. For FACS analyses, samples were analyzed on aFACSCanto II Flow Cytometer (Becton Dickinson, Franklin Lakes, N.J.).1×10⁸ events, which corresponded to individual Aph organisms and hostcellular debris, were collected in the VCU Flow Cytometry and ImagingShared Resource Facility. Post data-acquisition analyses were performedusing the FCS Express 4 Flow Cytometry software package (De NovoSoftware, Los Angeles, Calif.).

In Silica Analyses.

The MEMSAT-SVM algorithm (bioinf.cs.ucl.ac.uk/psipred) was used topredict the membrane topology of Aph OmpA. Predicted signal sequencesfor Anaplasma spp., Ehrlichia spp., and O. tsutsugamushi OmpA proteinswere determined using TMPred (www.ch.embnet.org/software/TMPRED_form).Alignments of OmpA sequences (minus the predicted signal sequences) weregenerated using CLUSTAL W. The tertiary structure for Aph OmpA waspredicted using the PHYRE² (Protein Homology/analogy Recognition Engine,version 2.0) server (see the website at sbg.bio.ic.ac.uk/phyre2). Toassess how OmpA potentially interacts with sLe^(x), the OmpA tertiarystructure predicted by PHYRE² was docked with the crystal structure forsLe^(x) using the autodock vina algorithm.

Assay for Inhibition of Aph Binding and Infection.

For antibody blocking studies, infection assays were performed asdescribed, except that host cell-free Aph organisms were incubated withheat-killed mouse polyclonal antiserum targeting GST, GST-Asp14, orGST-OmpA (10-200 ug/ml) or rabbit polyclonal anti-OmpA (targeting OmpAaa23-40, aa43-58, or aa59-74) and/or anti-Asp14 peptide serum (targetingAsp14 aa98-112 or aa 113-124) for 30 min, after which the bacteria wereadded to HL-60 cells in the continued presence of antiserum for 1 h.Unbound bacteria were removed and aliquots of host cells were examinedfor bound Aph organisms using indirect immunofluorescence microscopy.The remainders of the samples were incubated for 48 h, after which hostcells were examined for the presence of morulae using indirectimmunofluorescence microscopy. For recombinant protein blocking studies,RF/6A or HL-60 cells were incubated with 4 μM GST; GST-Asp14; GST-OmpAorGST APH_1387_(Δ1-111) at 37° C. for 1 h. Host cells were washed with PBSto remove unbound proteins, fixed with paraformaldehyde for 1 h, andpermeabilzed with ice-cold methanol for 30 min. Protein binding to hostcells was assessed by indirect immunofluorescence microscopy usingrabbit anti-GST antibody (Invitrogen). For blocking studies, host cellswere incubated with recombinant proteins for 1 h after which Aphorganisms were added for an additional 24 h. Unbound bacteria wereremoved and the samples were incubated for 48 h followed byimmunofluorescence microscopy analysis for the presence of morulae.

Statistical Analyses.

The Student's t test (paired) performed using the Prism 4.0 softwarepackage (Graphpad; San Diego, Calif.) was used to assess statisticalsignificance. Statistical significance was set at p<0.05.

Example 1. Neutravidin Affinity Purification of Biotinylated Aph DCSurface Proteins and Two-Dimensional-Liquid Chromatography Tandem MassSpectrometry (2D-LC/MS-MS) Proteome Analysis Identifies Novel OuterMembrane Protein Candidates

DC bacteria were purified to remove the majority of contaminating hostcellular debris. DC surface proteins labeled by Sulfo-NHS-SS-Biotin wererecovered by neutravidin affinity chromatography (data not shown).Aliquots of input host cell-free DC lysate, affinity-captured DC surfaceproteins, neutravidin beads plus unlabeled DC whole cell lysate (lane3), and neutravidin beads alone were resolved by SDS-PAGE followed bysilver staining.

Because the Aph DC is the adherent and infectious form and thecomplement of DC surface proteins is unknown, we set out to identify DCsurface proteins. Aph infected HL-60 cells were sonicated to liberatethe bacteria from host cells and destroy fragile RC organisms. Electronmicroscopic examination of sonicated samples confirmed the presence ofDC, but not RC bacteria, along with host cellular debris (data notshown). DC organisms were surface-labeled and biotinylated proteins werecaptured by chromatography. Aliquots of affinity-captured DC proteins,input host cell-free DC lysate, neutravidin beads plus unlabeled DCwhole cell lysate, and neutravidin beads alone were resolved by SDS-PAGEfollowed by silver staining (data not shown). Comparison of the bandingpatterns of the input lysate and eluate revealed enrichment for manyproteins. With the exception of proteins of 44 kDa and 70 kDa, both ofwhich were recovered in low abundances, non-biotinylated DC whole celllysate proteins did not bind to neutravidin beads.

Eluted proteins were subjected to 2D-LC/MS-MS proteomic analysis.Resulting data were searched against 2 Aph-specific FASTA databases(RefSeq and Uniprot sources) using Protein Lynx Global Surveyor (PLGS)software. Table 5 summarizes a total of 56 identified Aph proteins, 47of which were identified in both the RefSeq and UnitProt sources.

TABLE 5 Aph DC proteins recovered post-surface labeling and affinitychromatography analyzed by 2D-nanoLC/tandem MS protein analysis TABLE 5.A. phagocytophilum DC proteins recovered post-surface labeling andaffinity chromatography analyzed by 2D-nanoLC/tandem MS protein analysisRefSeq^(a) UniProt^(b) Cov- Cov- mW^(c) Pep- erage Amount Pep- erageAmount Locus JCVI Description (Da) pI^(d) Score tides (%) (fmol)^(e)Score tides (%) (fmol) APH_1221 ^(f) P44 18ES outer membrane 45,799 5.620,608.4 131 78.0 269.0  20,363.3 133 78.0 222.1 protein expressionlocus with P44-18 APH_1287 P44 32 outer membrane 44,350 5.4 19,848.5 13773.9 343.3  19,451.2 137 75.1 375.9 protein APH_1229 P44 2b outermembrane 44,884 5.2 18,321.9 138 81.4  29.1 17,898.4 135 76.5 31.7protein APH_1169 P44 19 outer membrane 33,033 5.3 18,185.8 62 82.3524.8  17,902.6 65 82.3 633.6 protein APH_1269 P44 16 outer membrane45,261 5.6 16,839.7 114 69.0 314.4  16,779.1 116 69.0 550.3 proteinAPH_1275 P44 16b outer membrane 45,194 5.9 16,695.3 122 78.0  44.416,427.1 122 78.0 37.5 protein APH_1215 P44 14 outer membrane 46,133 5.413,580.3 129 77.1 788.0  13,490.5 123 76.7 664.6 protein APH_0172 P44outer membrane protein 7,236 4.4 11,994.3 18 94.0  0^(g) 11,807.8 1894.0 0 C terminal fragment APH_1235 Hypothetical protein 14,762 5.34,190.9 33 91.8 189.4  4,998.2 30 97.0 189.4 APH_0240 Chaperonin GroEL58,263 5.0 1,436.7 69 68.7  76.9 1,403.2 64 71.6 76.9 APH_0494 F0F1 ATPsynthase subunit 51,478 4.8 641.4 32 58.9  40.8 628.2 30 70.1 40.8 betaAPH_0405 Asp62 outer membrane 57,538 9.5 489.5 27 45.5  84.9 471.9 2138.6 106.4 protein APH_1087 Putative competence 26,084 4.8 458.2 10 36.9 32.9 519.9 10 36.9 32.9 lipoprotein ComL APH_1032 Elongation factor Tu42,831 5.1 415.5 19 44.8   0.0 398.1 19 35.1 51.1 APH_1190 Putative ATPsynthase F0 B 18,837 5.9 415.5 2 14.4  31.7 458.5 10 47.9 31.7 subunitAPH_0404 Asp55 outer membrane 63,644 8.9 413.1 21 26.8  49.3 413.9 2225.8 49.3 protein APH_0397 30S ribosomal protein S2 32,118 9.2 406.4 1232.8  66.8 392.5 12 36.1 66.8 APH_0036 Co chaperone GrpE 22,646 5.8394.7 4 33.2   0.0 372.7 4 33.2 0 APH_1404 Type IV secretion system46,871 4.7 388.9 8 22.8  34.5 379.4 7 21.7 34.5 protein VirB10 APH_0346Chaperone protein DnaK 69,676 4.9 381.2 25 34.4 177.7  380.1 24 36.4177.7 APH_0248 Hypothetical protein (Asp14) 13,824 4.9 359.0 10 58.1 0APH_1049 Major surface protein 5 23,341 4.7 353.7 4 22.5 170.6  339.9 322.5 170.6 APH_1334 F0F1 ATP synthase subunit 54,068 5.3 312.1 30 34.8180.0  270.5 23 28.5 0 alpha APH_0051 Iron binding protein 37,317 5.2252.9 4 14.6 0 318.8 5 17.9 109.1 APH_0853 Hypothetical protein 10,8339.3 249.9 4 62.9 0 162.7 1 15.5 0 APH_0625 Immunogenic protein; 34,6535.9 229.0 6 28.6 0 207.9 5 28.6 0 membrane transporter APH_1050 Putativephosphate ABC 37,567 5.6 221.0 3 16.5 0 192.1 1 2.7 0 transporterperiplasmic phosphate binding protein APH_1246 Glutamine synthetase typeI 52,383 6.0 216.0 9 10.2 0 228.0 10 10.2 0 APH_1232 Citrate synthase I45,591 5.8 213.8 5 19.7 0 151.0 2 3.6 0 APH_0600 Thiamine biosynthesisprotein 61,522 6.0 203.3 4 11.0 0 206.0 4 13.5 0 ThiC APH_0059Phenylalanyl tRNA 39,277 6.5 197.0 7 14.0 0 180.0 8 11.4 0 synthetasealpha subunit APH_0555 Cysteinyl tRNA synthetase 51,774 5.8 192.8 5 18.60 197.2 4 16.0 0 APH_0794 Hypothetical protein 27,119 7.1 183.9 2 8.4 0164.8 1 4.2 0 APH_0740 AnlcA 131,081 6.1 182.8 11 7.2 0 189.2 13 8.2 0APH_1258 Fructose bisphosphate 32,685 6.7 182.0 5 9.2 0 193.7 4 9.2 0aldolase APH_1025 50S ribosomal protein L7 L12 14,122 4.8 181.5 2 23.9 0APH_1292 Cell division protein FtsZ 41,975 5.0 181.3 3 13.3 0 205.0 310.5 0 APH_1210 OMP85 family outer 85,652 8.5 173.9 7 8.3 0 165.5 6 5.70 membrane protein APH_0283 50S ribosomal protein L2 29,772 11.5 169.5 38.3 0 154.1 2 6.2 0 APH_0893 Heat shock protein 90 71,123 4.9 167.9 612.7 0 173.7 9 17.0 0 APH_0111 Uridylate kinase 26,347 6.9 164.4 2 13.10 176.4 3 18.0 0 APH_0608 PpiC parvulin rotamase 67,363 4.9 161.4 1013.1 0 144.2 8 9.0 0 family protein APH_1359 Major outer membrane 31,6179.0 157.8 2 5.5 0 142.4 2 5.5 0 protein OMP-1A APH_1084 Cytochrome coxidase subunit 29,873 6.1 155.0 3 13.0 0 II APH_0422 Acetylglutamatekinase 35,726 4.6 151.9 2 7.0 0 APH_0971 Putative trigger factor 49,3584.8 140.8 3 13.0 0 138.3 2 10.0 0 APH_0038 CTP synthetase 59,416 5.5139.6 2 5.9 0 136.9 2 5.9 0 APH_1355 P44 79 outer membrane 50,321 8.7139.0 2 3.9 0 147.7 2 4.6 0 protein APH_0669 Bifunctional proline114,508 5.1 139.0 4 6.9 0 159.1 5 7.6 0 dehydrogenase pyrroline 5carboxylate dehydrogenase APH_0450 ATP dependent Clp protease 86,715 6.2138.0 2 1.6 0 ATP binding subunit ClpA APH_0231 Leucyl aminopeptidase54,611 5.5 128.8 3 11.4 0 APH_0874 Hypothetical protein 115,420 6.6123.2 5 2.9 0 APH_1017 Outer membrane protein 46,971 8.4 131.9 2 3.6 0Msp2 family APH_1339 Conserved domain protein 47,356 7.3 128.6 2 5.1 0APH_0168 Heme exporter protein CcmC 26,310 9.5 126.7 4 6.9 0 APH_0502tRNA pseudouridine synthase 28,012 8.8 131.9 2 3.6 0 A ^(a)Refseq, A.phagocytoplrilum, Downloaded February 2010 ^(b)UniProt, A.phagocytaphilum, Downloaded February 2010 ^(c)mW, molecular weight inDaltons ^(d)pI, isoelectric point ^(d)fmol, femtomoles ^(f)Proteins thathave been previously confirmed to be on the A. phagocytophilum surfaceand/or were recovered by surface biotinylation and affinitychromatography in the study by Ge and colleagues are denoted by boldtext. ^(g)Peptides that are considered in-source fragments are given a 0fmol value as their quantification is confounded by signal lost withinthe mass spectrometer.

All proteins for which at least two peptides were identified from eitherRefSeq or UnitProt and scored above a 5% false-discovery cutoff arelisted. Three protein identifications from each search result are likelyfalse-positives, and are most probably among those found on one searchresult. Nine proteins had previously been delineated as beingsurface-localized, thereby validating the efficacy of our approach. Tenparalogs of the major surface protein 2 [Msp2 (P44)] family wereidentified, eight of which yielded the highest PLGS scores.

Example 2. Selection of Aph OMP Candidates for Further Study. FIG. 1AIllustrates the Experimental Timeline Relative to the Infection Cycleand Stages of Aph Organisms During Infection of a Host

DC organisms were used to synchronously infect HL-60 cells and theinfection proceeded for 36 h, a time period that allows for the bacteriato complete their biphasic developmental cycle and reinitiate infection.Total RNA was isolated from the DC inoculum and from infected host cellsat several postinfection time points. RT-qPCR was performed usinggene-specific primers. Relative transcript levels for each target werenormalized to Aph 16S rRNA gene transcript levels using the 2^(−ΔΔC)_(T) method. To determine the relative transcription of OMP candidategenes between RC and DC organisms, normalized transcript levels of eachgene per time point (shown in FIG. 1B-D) were calculated as thefold-change in expression relative to expression at 16 h (encircled inthe experimental timeline in FIG. 1A), a time point at which the Aphpopulation consists exclusively of RC organisms. (FIG. 1A) Diagram ofthe experimental design highlighting the time points at which RNA wasisolated, the Aph biphasic developmental and infection stages, and theexpression categories into which each gene of interest was classifiedbased on its expression profile. (FIGS. 1B-D) RT-qPCR results for eachOMP candidate-encoding gene of interest are grouped as (1B) early stage,(1C) mid stage, and (1D) late stage depending on when during the courseof infection they are most highly expressed. (FIG. 1E) RT-qPCR resultsfor control genes. The data in FIGS. 1B-E are the means and standarddeviations of results for triplicate samples and are representative oftwo independent experiments that yielded similar results.

Several proteins were selected for differential gene expression analysisover the course of Aph infection. Asp14, APH_0625, and APH_0874 werechosen because they were hitherto hypothetical proteins. For theremainder of this paper, we will refer to “hypothetical” proteins forwhich we have demonstrated expression as “uncharacterized” proteins.APH_1049 (Msp5), APH_1210 (Omp85), and APH_1359 (Omp-1A) were selectedbecause, even though they are confirmed Anaplasma spp. proteins, theirdifferential gene expression patterns have yet to be studied. APH_0240(chaperonin GroEL), APH_0346 (DnaK), and APH_1032 (elongation factor Tu)were chosen because, even though these proteins play housekeeping roles,they have also been identified as surface proteins of Aph and otherbacterial species and/or have been linked to bacterial adhesion.

A limitation of the surface biotinylation-affinity proteomics method isthat it will not identify surface proteins that are inaccessible to thecross-linker, either due to a lack of free amine groups forcross-linking or due to excessive distance from the bacterial surface towhich it extends relative to the length of the cross-linker. Also,detergents may not fully extract integral membrane proteins or proteincomplexes. Lastly, a surface protein that is in low abundance may not bein sufficient quantity to be detected even if biotinylated. Werationalized that Aph genes upregulated during colonization of mammalianversus tick cells are important for infection of mammalian cells.Therefore, as a complementary approach, we selected 9 candidate genesthat are known to be preferentially expressed during infection of HL-60cells and endothelial cells versus infection of ISE6 (immortalized I.scapularis embryonic) cells and are predicted by the CELLO subcellularprediction server to localize to the Aph outer membrane. Thesecandidates, which were not detected by our or a previous surfaceproteomics study, are OmpA (homologous to peptidoglycan-associatedlipoprotein [Pal]; conserved among most Gram-negative bacteria),APH_1220 (Omp-1N), APH_1325 (Msp2), APH_0838, APH_0839, APH_0906,APH_0915, APH_1378, and APH_1412. We also selected aph_0441 andaph_1170, because they encode previously detected, but uncharacterizedAph surface proteins. The SignalP 3.0 server predicts 9 of the 20candidates—OmpA, Omp-1a, Omp-1N, Omp85, Msp2, Msp5, APH_0441, APH_0915,and APH_1378—to carry N-terminal signal peptide sequences. The TMPredalgorithm (see the website at ch.embnet.org/software/TMPRED_form.html)predicts that all candidates except for Asp14 and APH_1412 carry one ormore transmembrane domains.

Example 3. Differential Transcription Profiling of Omp Candidate GenesThroughout the Aph Infection Cycle

To gain insight into the transcription of the 20 genes of interestduring the Aph infection cycle, we synchronously infected HL-60 cellswith DC organisms and allowed the infection to proceed in order for thebacteria to complete their biphasic developmental cycle and initiate asecond round of infection. We isolated total RNA from DC organisms usedas the inoculum and from bacteria recovered at several post-infectiontime points. RT-qPCR was performed on total RNA using gene-specificprimers. Relative transcript levels for each target were normalized toAph 16S rRNA gene (aph_1000) transcript levels using the 2^(−ΔΔC) _(T)method. To facilitate identification of genes that are up-regulated inthe infectious DC form compared to the non-infectious RC form,normalized transcript levels for each gene per time point werecalculated as the fold-change in expression relative to expression at 16h, a time point at which the Aph population consists exclusively of RCorganisms.

Genes of interest were classified as early (0-12 h), mid (12-24 h), orlate stage (24-36 h) (FIG. 1A). The early stage correlates with DCadhesion and invasion, DC to RC differentiation, and initiation of RCreplication. Early stage gene transcription increased at 4 h and peakedat 8 h or 12 h, except for asp14 and aph_0346, both of which peaked at 4h (FIG. 1B). Expression levels of all early stage genes began toincrease again between 28 and 36 h, which correspond to the periodduring which Aph RC organisms differentiate to DC organisms and initiatethe second round of infection. Mid stage gene expression, whichcoincides with a period of extensive Aph replication, peaked at 16 h(FIG. 1C). Late stage genes were upregulated between 24 and 36 h (FIG.1D), a period that correlates with the conversion of RC to DC organisms,DC exit, and initiation of the second round of infection. All targetmRNAs were detected in host cell-free DC organisms (FIG. 1). Transcriptlevels of asp14, aph_0346, aph_0838, aph_0839, aph_0874, aph_0915,aph_1378, aph_1412, and msp2 were more abundant in DC bacteria used asthe inoculum than in RC bacteria at 16 h. Because msp2 (P44), asp62, andasp55 encode confirmed Aph surface proteins and because the latter twoconstitute an operon, these genes were analyzed as controls. Coincidentwith the kinetics of the infection cycle, msp2 (p44) transcriptionsteadily increased from 4 to 28 h, after which it pronouncedly declinedby 32 h. The transcriptional profiles of asp55 and asp62 were highlysimilar, which reinforces the accuracy of the expression data obtainedfor all genes.

Example 4. Aph Transcriptionally Upregulates OmpA and Asp14 DuringBinding and Invasion of Myeloid but not Endothelial Cells

It takes up to four hours for the majority of bound Aph organisms toenter and reside within nascent host cell-derived vacuoles. Thus, genesthat are upregulated between 0 and 4 h and in the initial hoursfollowing bacterial entry conceivably encode products that are importantfor invasion and/or establishing infection. Of all genes examined, asp14is the most abundantly expressed at 4 h (FIG. 1B-E), and asp14 and ompAexhibit the most abundant non-DC to RC-normalized transcript levels(data not shown). Accordingly, we more closely examined the expressionprofiles of ompA and asp14. Differential expression analyses of ompA andasp14 during Aph invasion of HL-60 and RF/6A cells, during Aph bindingto PSGL-1 CHO cells, and during transmission feeding of Aph infected I.scapularis ticks is shown in FIG. 2A-C. Aph organisms were incubatedwith HL-60 (2A), RF/6A (2B), and PSGL-1 CHO cells (2C) for 4 h, a periodthat is required for bacterial adherence and for ≥90% of bound bacteriato invade host cells. Aph cannot invade PSGL-1 CHO cells. Total RNA wasisolated from the DC inoculum and from host cells at 1, 2, 3, and 4 hpost-bacterial addition. (2D) Aph infected I. scapularis nymphs wereallowed to feed on mice for 72 h. Total RNA was isolated from thesalivary glands of uninfected and transmission fed ticks that had beenremoved at 24, 48, and 72 h post-attachment. Total RNA was isolated fromcombined salivary glands and midguts from unfed ticks. (2A-2D) RT-qPCRwas performed using gene-specific primers. Relative transcript levelsfor asp14 and ompA were normalized to Aph 16S rRNA gene transcriptlevels. The normalized values in FIGS. 2A-C are presented relative toasp14 or ompA transcript levels of the DC inoculum. Data are the meansand standard deviations of results for triplicate samples and arerepresentative of two independent experiments that yielded similarresults.

Aph DC bacteria were added to HL-60 and RF/6A cells, after which RT-qPCRwas performed on total RNA isolated at 1, 2, 3, and 4 h. RNA isolatedfrom the DC bacterial inoculum served as a reference control. asp14 wasupregulated at all time points during adhesion and invasion of HL-60cells and exhibited a maximal increase at 2 h, whereas ompA demonstrateda maximal increase at 4 h (FIG. 2A). Neither ompA nor asp14 wasupregulated during binding and invasion of endothelial cells (FIG. 2B).

Example 5. Aph Engagement of PSGL-1 Promotes Upregulation of Asp14, butnot OmpA

We next examined whether Aph binding to PSGL-1 upregulates either asp14or ompA. Chinese hamster ovary cells transfected to express PSGL-1(PSGL-1 CHO cells) are ideal models for studying Aph-PSGL-1 interactionsbecause they support Aph binding, while untransfected CHO cells thatlack PSGL-1 expression do not. Thus, Aph binding to PSGL-1 CHO cellsoccurs exclusively through bacterial engagement of PSGL-1. DC bacterialbinding to PSGL-1 CHO cells upregulated asp14, but not ompA (FIG. 2C).

Example 6. Aph Upregulates OmpA and Asp14 During I. scapularisTransmission Feeding

Aph genes that are induced during the bloodmeal of infected I.scapularis ticks are presumably important for establishing infection inmammals. We examined ompA and asp14 expression in Aph infected I.scapularis nymphs during transmission feeding on naïve mice. Transcriptsfor neither ompA nor asp14 were detected in unfed Aph infected nymphs(FIG. 2D). Both asp14 and ompA were induced during transmission feeding,being first detected at 24 h and 48 h, respectively.

Example 7. Aph Expresses OmpA and Asp14 During Infection of HL-60 Cellsand During Murine and Human Infection

As illustrated in FIGS. 3A and B, whole cell lysates of E. coli (U), E.coli induced (I) to express GST-OmpA (FIG. 3A) or GST-Asp14 (FIG. 3B),and GST-OmpA (3A) or GST-Asp14 (3B) purified (P) by glutathionesepharose affinity chromatography were separated by SDS-PAGE and stainedwith Coomassie blue. (FIGS. 3C and D) Western blot analyses in whichmouse anti-OmpA (αOmpA; raised against GST-OmpA) and αAsp14 (raisedagainst GST-Asp14) were used to screen whole cell lysates of uninfectedHL-60 cells and Ap organisms. The blot in FIG. 3D was stripped andrescreened with anti-Msp2 (P44) (αP44). The thin and thick arrows denoteAsp14 and Msp2 (P44), respectively. (FIG. 3E) Western blotted MBP-P44,MBP, and whole cell lysates of uninfected HL-60 cells and Aph organismswere screened with αAsp14. The blot was stripped and rescreened withanti-MBP-P44. (FIG. 3F) GST-Asp14 was resolved by SDS-PAGE undernon-reducing and reducing conditions, Western-blotted, and screened withαAsp14. (FIG. 3G) Western-blotted GST-OmpA, GST-Asp14, and GST werescreened with sera from an HGA patient and an experimentally infectedmouse.

The coding regions of ompA (excluding the signal sequence; 19.9 kDa) andasp14 (13.8 kDa) were cloned and expressed in E. coli as N-terminalglutathione-S-transferase (GST)-tagged fusion proteins designated asGST-OmpA and GST-Asp14, respectively (FIGS. 3A and B). Afterglutathione-Sepharose affinity chromatography, purified GST-OmpA andGST-Asp14 appeared as 46.0- and 39.8-kDa bands, respectively, uponSDS-PAGE. Each fusion protein was used to immunize mice. Polyclonalanti-OmpA antisera recognized proteins of 22.1 kDa and 19.9 kDa, whichcorrespond to OmpA preprotein and mature OmpA, respectively, in an Aphlysate but not an uninfected HL-60 cell lysate (FIG. 3C). In addition tothe anticipated 13.8 kDa band, anti-Asp14 detected a band ofapproximately 42 kDa in a lysate of Aph, but not uninfected HL-60 cells(FIG. 3E). Anti-Asp14 occasionally detected another band ofapproximately 28 kDa on blots of Aph lysates (data not shown). Eventhough the 42-kDa band is close in size to that anticipated for Msp2(P44), anti-Asp14 failed to recognize Aph-derived maltose bindingprotein (MBP)-tagged Msp2 (P44) (FIGS. 3D and E). An amino acid sequencealignment of Asp14 with Msp2 (P44)-23, the most abundantly expressedMsp2 (P44) paralog of the Aph NCH-1 strain [56,57], revealed noconsiderable stretches of homology (data not shown). GST-Asp14multimerizes when fractionated by non-denaturing SDS-PAGE (FIG. 3F).Thus, the 28- and 42-kDa bands in the Aph lysate recognized byanti-Asp14 are presumably multimeric complexes that consist exclusivelyof or contain Asp14. HGA patient serum and Aph infected mouse serumrecognize GST-OmpA and GST-Asp14 (FIG. 3G), signifying that Aphexpresses OmpA and Asp14 during human and murine infection.

Example 8. OmpA is Differentially Expressed by Aph During Infection ofMammalian Versus Tick Cells, while Asp14 is Expressed During Infectionof Both Mammalian and Tick Cells

Because Aph infects myeloid cells, endothelial cells, and I. scapulariscells in vivo and in vitro, we examined Asp14 and OmpA expression duringinfection of HL-60 cells, RF/6A cells, and ISE6 cells, (data not shown).Aph infected HL-60, RF/6A, and ISE6 cells were fixed and viewed byconfocal microscopy to determine immunoreactivity with antibodiesagainst Msp2 (P44) (major surface protein; used to identify bacteria),OmpA, or Asp62 (confirmed surface protein). Both OmpA and Asp62 stainingyield comparable ring-like bacterial surface staining patterns. Resultsdescribed are the means and standard deviations of results of at leasttwo separate experiments. At least 200 Msp2 (P44)-positive morulae werescored for Asp14 and OmpA per condition. Confocal microscopicexamination using anti-Asp14 or anti-OmpA in conjunction with antiserumagainst constitutively expressed Msp2 (P44) revealed that 100.0% ofmorulae (intravacuolar Aph colonies) in each of the three cell lines wasAsp14-positive. OmpA was detected in 100.0% and 48.6±15.9% of moruale inHL-60 and RF/6A cells, respectively, but was detected in only 7.0±3.5%of morulae in ISE6 cells (results were statistically significant,p<0.001). Anti-OmpA binding to intracellular Aph organisms yielded aring-like staining pattern on the periphery of each bacterium thatoverlapped with signal corresponding to the confirmed surface protein,Msp2 (P44)(data not shown). The anti-OmpA staining pattern was similarto that of another confirmed Aph surface protein, Asp62. Anti-Asp14staining was more uniformly distributed over the bacterial cells andexhibited partial overlap with Msp2 (P44) (data not shown).

Example 9. Surface Localization of OmpA and Asp14

To assess surface presentation of OmpA and Asp14, intact Aph DCorganisms were incubated with trypsin followed by solubilization,western blotting, and screening with anti-OmpA or anti-Asp14 todetermine if immunoaccessible domains of either target protein arepresented on the bacterial surface, shown in FIGS. 4A and B. In FIG. 4A,Intact DC bacteria were incubated with trypsin or vehicle control, lysedin RIPA buffer, fractionated by SDS-PAGE, and immunoblotted. Westernblots were screened with antisera targeting OmpA, Asp55, Msp5, Asp14,Msp2 (P44), or APH_0032. Data are representative of two experiments withsimilar results. In FIG. 4B, Transgenic Aph organisms expressing GFPwere incubated with preimmune mouse serum, mouse anti-Asp14 oranti-OmpA, or serum recovered from an Aph infected mouse. Primaryantibodies were detected with anti-mouse IgG conjugated to Alexa fluor647. Flow cytometry was used to determine the percentage of Alexa fluor647- and GFP-positive DC organisms per sample. The fold-increases in thepercentages of Alexa fluor 647-positive, GFP-positive DC organisms foreach sample relative to preimmune serum are provided. Results presentedare the means±SD of three experiments. Statistically significant (*,p<0.05) values are indicated. Positive control antisera targeted Asp55,Msp2 (P44), and Msp5. Negative control antiserum was specific forAPH_0032, which is an Aph effector and is not a surface protein.Anti-Asp55 is specific for a peptide epitope of a surface-exposed loopof the target protein. Considerably less detection of Asp55, OmpA,Asp14, and Msp5 was observed for trypsin-treated than for vehiclecontrol-treated bacteria, whereas Msp2 (P44) signal intensity waspartially reduced and no loss in APH_0032 signal resulted (FIG. 4A). Asa complementary approach to verify surface presentation of OmpA andAsp14, transgenic Aph DC organisms expressing GFP were recovered fromsonicated HL-60 cells and screened with anti-OmpA, anti-Asp14, orcontrol antisera using flow cytometry. Serum from an Aph infected mouserecognized 1.9±0.8-fold more organisms than preimmune mouse serum (FIG.4B). Anti-OmpA and anti-Asp14 recognized 5.0±2.9- and 4.9±2.7-fold moreAph organisms expressing GFP than preimmune mouse serum (FIG. 4B).

Example 10. Pretreatment of Aph with Anti-OmpA Reduces Infection ofHL-60 Cells

Because OmpA is exposed on the Aph surface, we determined if treating DCorganisms with heat-inactivated anti-OmpA serum prior to incubation withHL-60 cells alters bacterial adhesion to or infection of host cells.Anti-OmpA had no effect on bacterial adhesion, but significantly reducedinfection (FIG. 5A-D). Pretreatment of bacteria with mouse polyclonalanti-GST serum had no effect on binding or infection.

Example 11. In Silico Analyses of Aph OmpA and Comparisons with Homologsfrom Other Anaplasmataceae Pathogens

Since anti-OmpA inhibits Aph infection, we hypothesized that OmpA maycontribute to infection of host cells. We performed in silico analysesto identify the predicted extracellular region of OmpA, which wouldputatively contain any receptor-binding domain, and to assess whetherthis and other regions of OmpA are conserved among its homologs fromother Rickettsiales bacteria. The OmpA N-terminal region extendingthrough to amino acid 86 is predicted to comprise the only extracellulardomain, and amino acids 87-102 are predicted to form a transmembranehelix (FIG. 6A). A multiple sequence alignment revealed that the AphOmpA sequence has several shaded stretches that exhibit identity orsimilarity with its homologs from other Anaplasma spp. and Ehrlichiaspp. (FIG. 6A).

The PHYRE² server (see the website at sbg.bio.ic.ac.uk/phyre2) predictstertiary structures for protein sequences and threads the predictedstructures on known crystal structures. The highest scoring model forAph OmpA that exhibits the greatest amino acid sequence identity withthe crystal structure on which it was threaded, Bacillus chorismateOmpA, is presented in FIG. 6B. Amino acids 44-56 are predicted to form asurface-exposed helix and loop, as indicated by arrows. The peptideK[IV]YFDaxK (where “a” and “x” represent a non-polar and any amino acid,respectively), that corresponds to Aph OmpA residues 49-56 is conservedamong Anaplasma spp. and Ehrlichia spp. OmpA proteins.

Example 12. Interactions of GST-OmpA with Endothelial Cells

We tested if we could detect GST-OmpA binding to RF/6A cells. Since OmpAproteins of Aph and O. tsutsugamushi exhibit regions of identity, O.tsutsugamushi infects endothelial cells, and it is unknown whether O.tsutsugamushi OmpA interacts with endothelial cells, we also assessedwhether GST-tagged O. tsutsugamushi OmpA (GST-OtOmpA) bound to RF/6Acells. Negative controls for cellular adhesion were GST alone andGST-tagged APH_1387 amino acids 112-579 (GST-APH_1387₁₁₂₋₅₇₉). APH_1387is an Aph effector that associates with the bacterium's vacuolarmembrane. APH_1387 amino acids 112-579 lack the transmembrane domainthat is required for interacting with eukaryotic cell membranes(unpublished observation). GST-OmpA but not GST bound to RF/6A cells(data not shown). Neither GST-APH_1387₁₁₂₋₅₇₉ nor GST-OtOmpA bound thehost cells. GST-tagged Aph OmpA binding to RF/6A cells is thereforespecific because recombinant form of neither an irrelevant Aph proteinnor OmpA derived from another Rickettsiales bacterium binds to RF/6Acells. GST-OmpA binding to RF/6A cells does not involve PSGL-1 orsLe^(x) since antibodies targeting either receptor fail to bind RF/6Acells (data not shown) and a previous report demonstrated thatendothelial cells do not express PSGL-1. We examined if preincubatingRF/6A cells with GST-OmpA competitively inhibits Aph binding orinfection. GST-OmpA but not GST significantly inhibited infection (datanot shown). Neither recombinant protein inhibited Aph adhesion (data notshown).

Example 13. Sialidase and Trypsin Treatments Markedly Reduce GST-OmpABinding to Host Cells

Enzymatic removal of sialic acid residues from myeloid cell surfacespronouncedly inhibits Aph binding and infection. Sialic acid residuesare also important for Aph infection of RF/6A cells, as pretreatment ofRF/6A cells with sialidases reduced Aph infection by 52.8±1.4% (data notshown). The MAL-II lectin recognizes sialic acids that are attached togalactose units via α2,3-linkages. The SNA lectin preferentially bindsto sialic acid attached to galactose in an α2,6-linkage. Sialidasetreatment abolished MAL-II binding and markedly reduced SNA binding,indicating that the sialidase cocktail completely removed α2,3-linkedsialic acids and partially removed α2,6-linked sialic acids. GST-OmpAdid not bind as well to RF/6A cells that had been incubated in thevehicle control buffer as compared to other buffers. Nonetheless,GST-OmpA binding to sialidase-treated cells was reduced. These resultssuggest that OmpA recognizes α2,3-linked sialic acids but is alsocapable of interacting with α2,6-linked sialic acids. Pretreatment ofRF/6A cells with trypsin, which would effectively digest protein andglycoprotein receptors, including terminally sialylated glycoproteins,nearly eliminated GST-OmpA binding.

Example 14. GST-OmpA Competitively Inhibits Aph Infection of HL-60 Cells

To define the relevance of OmpA to Aph infection of human myeloid cellsand to delineate the OmpA region that is critical for cellular invasion,we examined if preincubating HL-60 cells with GST-OmpA or fragmentsthereof inhibits infection by Aph DC organisms. GST-tagged full-lengthOmpA and OmpA₁₉₋₇₄, which comprises the majority of the predictedextracellular domain, but not GST-OmpA₇₅₋₂₀₅ or GST alone had no effecton adhesion (data not shown), but significantly inhibited infection(FIGS. 7A and B).

Example 15. GST-OmpA Inhibits Aph Binding to sLe^(x)-Capped PSGL-1

Aph binding to the α2,3-linked sialic acid determinant of sLe^(x) isnecessary for the bacterium to optimally engage sLe^(x)-capped PSGL-1and leads to infection of myeloid cells. Since GST-OmpA recognizesα2,3-sialic acid and competitively inhibits Aph infection of HL-60cells, we rationalized that GST-OmpA binds to α2,3-sialic acid ofsLe^(x). To test this, we incubated PSGL-1 CHO cells with GST-OmpA in anattempt to block Aph access to the α2,3-sialic acid determinant ofsLe^(x)-capped PSGL-1 and thereby inhibit bacterial adherence to thesecells. As a positive control for preventing bacterial access to theα2,3-linked sialic acid determinant of sLe^(x), PSGL-1 CHO cells wereincubated with CSLEX1. PSGL-1 CHO cells treated with GST or mouse IgMserved as negative blocking controls. GST-OmpA reduced Aph binding tosLe^(x)-modified PSGL-1 by approximately 60% relative to GST alone, andthis degree of inhibition was comparable to the blocking afforded byCSLEX1 (data not shown).

Example 16. Model for how Aph OmpA Interacts with its Receptor toPromote Infection of Host Cells (FIG. 8A-D)

Sialic acid has long been known to be a determinant that is importantfor Aph infection. This study demonstrates that OmpA targets sialylatedglycoproteins to promote Aph infection. Our results fit the model thatAph employs multiple surface proteins to bind three determinants ofsLe^(x)-capped PSGL-1 to infect myeloid cells (FIG. 8A). When these dataare examined in the context of results obtained from our own studies andothers, the respective contributions of sialic acid, α1,3-fucose, andPSGL-1 N-terminal peptide to Aph binding and entry become clearer.Treating myeloid cells with CSLEX1 to block A. phagocytophilum bindingto the sialic acid determinant of sLe^(x) markedly reduces infection(FIG. 8C), a phenomenon that is analogous to the inhibitory action ofGST-OmpA. Moreover, the inhibitory effects of CSLEX1 and GST-OmpA on Aphbinding to PSGL-1 CHO cells are nearly identical. Therefore, while OmpAis capable of binding sialic acid determinants of varied sialylatedglycans, its specific interaction with the sialic acid residue ofsLe^(x) is important for bacterial entry. GST-OmpA and GST-OmpA₁₉₋₇₄binding to host cells reduces Aph infection of HL-60 cells byapproximately 52 and 57%, respectively, but has no inhibitory effect onbacterial adhesion. Thus, bacterial recognition of the PSGL-1N-terminus, α1,3-fucose of sLe^(x), and perhapssLe^(x)-/PSGL-1-independent interactions that still occur when theOmpA-sialic acid interaction is disrupted facilitate bacterial bindingbut lead to sub-optimal infection (FIG. 8B). Antibodies that blockaccess to the PSGL-1 N-terminal peptide determinant prevent bacterialbinding and infection. Therefore, the collective avidity mediated byOmpA interaction with sialic acid together with Aph recognition ofα1,3-fucose is insufficient to promote bacterial adhesion and,consequently, entry in the absence of PSGL-1 recognition (FIG. 8D).

Example 17. Pretreating Aph with Anti-Asp14 Inhibits Infection of HL-60Cells

Since Asp14 is a surface protein, we examined if incubating Aph DCorganisms with heat-inactivated Asp14 antiserum prior to adding them toHL-60 cells inhibited bacterial binding or infection. Anti-Asp14 had noeffect on Aph adhesion, but reduced infection by approximately 33% andlowered the mean number of morulae per cell by approximately 54%, (FIGS.9A-D). Inhibition was specific to Asp14 antiserum, as GST antiserum didnot alter bacterial binding or infection.

Example 18. The Asp14 C-Terminal Region Binds Mammalian Host Cells

Since Asp14 is an exposed outer membrane protein and anti-Asp14 reducesAph infection, we rationalized that Asp14 may interact with mammalianhost cell surfaces to promote infection. To test this possibility and toidentify the Asp14 region that is sufficient for optimal adherence, weexamined if GST-tagged Asp14 or portions thereof bind to RF/6A cells.GST alone and GST-tagged APH_1387 amino acids 112-579(GST-APH_1387₁₁₂₋₅₇₉) were negative controls. APH_1387 is an Aph proteinthat localizes to the pathogen's vacuolar membrane and does notassociate with the host cell surface. GST-Asp14 but neither GST norGST-APH_1387₁₁₂₋₅₇₉ bound to RF/6A cells (FIG. 9A-D). The binding domainis carried on the Asp14 C-terminal half, as GST-Asp14₆₅₋₁₂₄ but notGST-Asp14₁₋₆₄ exhibited binding. GST-Asp14₁₋₁₀₀ and GST-Asp14₁₋₁₁₂ wereunable to bind RF/6A cells (data not shown). Thus, Asp14 residues101-124 contain the minimal region that is sufficient to facilitateadhesion to mammalian cell surfaces.

Example 19. GST-Asp14 Requires Asp14 Residues 101-124 to CompetitivelyInhibit A. phagocytophilum Infection of Mammalian Host Cells

We next determined if GST-tagged Asp14 or fragments thereof couldinhibit A. phagocytophilum infection. GST-Asp14 and GST-Asp14₆₅₋₁₂₄ eachsignificantly reduced infection of HL-60 and RF/6A cells relative to GSTalone (FIG. 10A-D). GST-Asp14₁₋₁₀₀ and GST-Asp14₁₋₁₁₂ had no effect oninfection of HL-60 cells (FIGS. 10A and B). GST-Asp14₁₋₁₁₂ did not lowerthe percentage of infected RF/6A cells, but reduced the mean number ofmorulae per RF/6A cell comparably to GST-Asp14₆₅₋₁₂₄ (FIGS. 10C and D).Pretreating host cells with GST-Asp14 fusion proteins prior toincubation with bacteria failed to inhibit A. phagocytophilum binding(data not shown). Thus, A. phagocytophilum binding to mammalian hostcells is Asp14-independent, but Asp14 is important for bacterialinvasion.

Example 20. The Asp14 C-Terminus is Positively Charged and Residues101-115 Constitute a Conserved Domain Among Homologs from Anaplasma andEhrlichia Species

Based on our results, a domain that lies within Asp14 amino acids101-124 is involved in mediating interactions with host cells thatpromote A. phagocytophilum infection. To determine if this or any otherAsp14 region is conserved among Anaplasmataceae members, we aligned theprimary amino acid sequences of Asp14 with its homologs from two A.marginale strains and three monocytotropic Ehrlichia species. Doing soidentified two conserved regions, the first of which corresponds toAsp14 amino acids 19-61 (FIG. 11). The second conserved region alignswith Asp14 residues 101-115. The consensus sequence for this regionamong the Anaplasma and Ehrlichia spp. Asp14 homologs isL[RK]aIKKR[IL]LRLERxV, where “a” and “x” represent a non-polar and anyamino acid, respectively. Beginning at tyrosine 116, the Asp14C-terminus bears no sequence homology to its A. marginale and ehrlichiaecounterparts. The Asp14 C-terminus (amino acids 101-124) has a charge of+4.91 despite the entire protein sequence having a charge of −3.10. Asimilar trend is observed when the charges of the Asp14 homologs'C-termini and entire protein sequences are examined.

Example 21. GST-Asp14 and GST-OmpA Together More Pronouncedly Inhibit A.phagocytophilum Infection of HL-60 Cells than Either Protein Alone

We examined whether we could improve upon the protection against A.phagocytophilum infection afforded by GST-Asp14 or GST-OmpA bypretreating HL-60 cells with both recombinant proteins. Consistent withprevious results, 35.5±7.4% of GST-OmpA-treated and 53.2±11.8% ofGST-Asp14-treated HL-60 cells became infected (FIG. 11A). However, HL-60cells that had been preincubated with both GST-Asp14 and GST-OmpA werebetter protected against A. phagocytophilum infection, as only 9.9±9.4%of cells developed morulae. To prove that the synergistic reduction ininfection was specific to the combinatorial effect of GST-Asp14 andGST-OmpA and not simply due to the presence of excess recombinantprotein, we treated HL-60 cells with GST-Asp14 and GST-OmpA,GST-Asp14₁₋₁₀₀ (does not block infection; data not shown) and GST-OmpA,or GST-Asp14 and GST-OmpA₇₅₋₂₀₅ (does not block infection). HL-60 cellstreated with GST-Asp14₁₋₁₀₀ and GST-OmpA or GST-Asp14 and GST-OmpA₇₅₋₂₀₅exhibited reductions in infection and bacterial load comparable to cellstreated with GST-Asp14 or GST-OmpA alone (FIGS. 12A and B). HL-60 cellstreated with GST-Asp14 and GST-OmpA exhibited an approximate 4.5-foldreduction in the percentage of infected cells relative to cells treatedwith either GST-Asp14₁₋₁₀₀ and GST-OmpA or GST-Asp14 and GST-OmpA₇₅₋₂₀₅(FIG. 12A).

Example 22. Peptide Antisera Blocking Reveals that the OmpA InvasinDomain Lies within Amino Acids 59-74

We had rabbit antiserum raised against peptides corresponding to OmpAamino acids 23-40, 41-58, and 59-74. We confirmed by ELISA that eachserum is specific for recombinant OmpA and only the peptide againstwhich it was raised (FIG. 13A). Pretreating A. phagocytophilum withserum specific for OmpA₅₉₋₇₄ but neither of the other two peptide serasignificantly inhibited A. phagocytophilum infection of host cells invitro (FIG. 13B). Also, treatment of bacteria with OmpA₅₉₋₇₄ serum butnot OmpA₂₃₋₄₀ serum or OmpA₄₁₋₅₈ serum prevents A. phagocytophilumbinding to its known receptor, sialylated PSGL-1 (FIG. 13C).

Please note that even thought amino acids 59-74 are most important forOmpA to promote infection that amino acids 23-58 are predicted to bepresented on the A. phagocytophilum surface and could therefore be acomponent of a protective vaccine.

Example 23. Linker Insertions Disrupt the Ability of GST-OmpA toAntagonize A. phagocytophilum Infection of Mammalian Host Cells andSupport that the Invasin Domain Lies within Amino Acids 59-74

We also generated a series of glutathione-S-transferase (GST)-taggedOmpA proteins having an insertion of 5 amino acids (CLNHL) at definedlocations. The purpose of the insertion of the amino acid “linker” wasto disrupt any OmpA domain that facilitates binding of the protein tohost cell surfaces. Individual plasmids encoding GST-OmpA proteinscarrying linker insertions between aspartate 34 and leucine 35;isoleucine 54 and glycine 55; proline 62 and glycine 63; isoleucine 67and leucine 68; glutamate 72 and glutamine 73; or aspartate 77 andaspartate 78 were generated by PCR mutagenesis of the plasmid encodingGST-OmpA (FIG. 14). E. coli was transformed with each plasmid, inducedto express the GST-OmpA proteins, and the proteins were purified byglutathione affinity chromatography. Adding recombinant wild-type OmpAand several OmpA insertion mutant proteins to host cells successfullyinhibited A. phagocytophilum infection of host cells (FIG. 15). Thesedata indicate that the OmpA proteins were still able to bind to the OmpAreceptor and competitively inhibit bacterial access to the receptor.However, only the GST-OmpA protein bearing a linker insertion betweenisoleucine 67 and leucine 68 lost the ability to competitively inhibitinfection, which indicates that disruption of the region encompassed byamino acids 67 and 68 and its flanking amino acids abrogates the abilityof OmpA to bind its receptor.

Example 24. Alanine Substitution Experiments Identify that Amino Acidswithin OmpA Aa59-74 are Important for Infection

To identify specific amino acids that are important for OmpA to bind toand mediate infection of host cells, we performed PCR mutagenesis tocreate plasmids encoding GST-OmpA bearing single or double alaninesubstitutions at D53, K64, E69, K60A, K65, E72A, KK6065AA, KK6064AA,KKK606465AAA, or K64 and K65. The proteins were purified and added tomammalian host cells. Next, A. phagocytophilum bacteria were incubatedwith the host cells. GST-OmpA, GST-OmpAD53A, and GST-OmpA eachsignificantly inhibited infection whereas GST alone did not (FIG. 16).The abilities of GST-OmpAK64A and GST-OmpAKK6465AA to antagonizeinfection were significantly less than that of GST-OmpA, which indicatesthat OmpA amino acids 64 and 65 are important for OmpA to properly bindto host cells and for recombinant OmpA to serve as a competitive agonistagainst A. phagocytophilum infection]

Example 25. In Silico Modeling of OmpA Interactions with its Receptor

The tertiary structure for A. phagocytophilum OmpA was predicted usingthe PHYRE² (Protein Homology/analogy Recognition Engine, version 2.0)server (see the website at sbg.bio.ic.ac.uk/phyre2). The PHYRE² serverpredicts tertiary structures for protein sequences and threads thepredicted structures on known crystal structures. The highest scoringmodel predicts that amino acids 59-74 to be part of a surface-exposedhelix that would be available to interact with other molecules (data notshown). Indeed, when the autodock villa algorithm(http://vina.scripps.edu) is used to assess whether OmpA binds to itsknown receptor, sialic acid of the sialyl Lewis x antigen, the lowestfree energy models predict that Lysine 64 interacts with sialic acid(data not shown).

Example 26. The Asp14 Invasin Domain Lies within Amino Acids 113-124

The structure of Asp14 is not known and it cannot be predicted becauseit bears no semblance to any crystal structure. Next, we set out toidentify the region of Asp14 that is important for infection. We knewthat the Asp14 invasin domain lies within amino acids 101-124. We hadrabbit antiserum raised against peptides corresponding to Asp14 aminoacids 101-112 and 113-124. We confirmed by ELISA that each serum isspecific for recombinant Asp14 and only the peptide against which it wasraised (FIGS. 17A and B). Pretreating Aph with serum specific forAsp14₁₁₃₋₁₂₄ but not Asp14₁₀₁₋₁₁₂ significantly inhibited bacterialinfection of host cells in vitro (FIG. 18).

Example 27. Treating Aph with Antibodies Targeting OmpA Aa59-74 andAsp14 Aa113-124 Together Pronouncedly Inhibits Infection of MammalianHost Cells

Treating Aph organisms with anti-OmpA₅₉₋₇₄ or anti-Asp14₁₁₃₋₁₂₄significantly inhibits infection of mammalian host cells in vitro (FIG.19). Treating the bacteria with both anti-OmpA₅₉₋₇₄ and Asp14₁₁₃₋₁₂₄even more pronouncedly inhibits infection.

Example 28. Aph OmpA and A. marginale OmpA Share B-Cell Epitopes

A. marginale infects bovine red blood cells and costs the cattleindustry hundreds of millions of dollars annually. A. marginale OmpA andAph OmpA, though not identical, are very similar, including the regioncorresponding to Aph OmpA aa19-74 (SEQ ID NO:05). Therefore, a vaccinepreparation that includes SEQ ID NO:05, alone or in combination withother sequences of the invention is also effective in providingprotection against A. marginale infection. GST-tagged Aph OmpA,GST-tagged A. marginale OmpA (AM854), and GST alone were subjected toSDS-PAGE and transferred to nitrocellulose membrane. The blots werescreened with serum from a cow that had been infected with A. marginaleor with serum from a cow that had been immunized with purified A.marginale outer membrane proteins. Both sera recognized GST-tagged OmpAproteins not GST (data not shown), thereby demonstrating that OmpAproteins from Aph and A. marginale share B cell epitopes. Serum raisedagainst Aph OmpA amino acids 41-58 or 59-74 recognize GST-A. marginaleOmpA (AM854) in both Western blot (FIG. 20A) and ELISA (FIG. 20B).

Example 28 Immunizing Against OmpA and/or Asp14 Protects Mice fromTick-Mediated Aph Infection

C3H/HeJ mice (female, 4-6 weeks of age) are immunized with 10 ug ofGST-OmpA (full length), GST-OmpA₁₉₋₇₄, GST-Asp14; 10 ug each of GST-OmpAand GST-Asp14; or 10 ug each of GST-OmpA₁₉₋₇₄ and GST-Asp14 in CompleteFreund's Adjuvant. At two and four weeks following the initialimmunization, mice are boosted with the same amounts and combinations ofeach antigen in Incomplete Freund's Adjuvant.

Alternatively, C3H/HeJ mice are immunized with 50 ug of KLH-conjugatedpeptides corresponding to OmpA₂₃₋₄₀, OmPA₄₁₋₅₈, OmpA₅₉₋₇₄, Asp14₁₀₀₋₁₁₂,Asp14₁₁₃₋₁₂₄ and every possible combination thereof. The same adjuvantsand immunization schedule as in the preceding paragraph may be followed.

Five days following the second boost, aliquots of serum from each mouseare tested via ELISA to confirm that a humoral immune response wasmounted against OmpA, Asp14, and the respective portions thereof. At oneweek following the second boost, three Aph infected Ixodes scapularisticks are placed on each mouse and allowed to feed for 48 hours to allowfor transmission of the bacteria into the mice. On days 3, 8, and 12post tick feeding, peripheral blood is collected. DNA isolated from theblood is subjected to quantitative PCR using primers targeting the Aph16S rDNA and murine Beta-actin to determine the pathogen load in theperipheral blood (data not shown). This protocol is also useful whenadjuvants suitable for innocculating dogs, humans, or other mammals areused for respective species.

Example 29. Immunizing Against OmpA and/or Asp14 Protects Mice fromSyringe Inoculation of Aph Infection

C3H/HeJ mice (female, 4-6 weeks of age) are immunized with 10 ug ofGST-OmpA (full length), GST-OmpA₁₉₋₇₄, GST-Asp14; 10 ug each of GST-OmpAand GST-Asp14; or 10 ug each of GST-OmpA₁₉₋₇₄ and GST-Asp14 in CompleteFreund's Adjuvant. At two and four weeks following the initialimmunization, mice are boosted with the same amounts and combinations ofeach antigen in Incomplete Freund's Adjuvant.

Alternatively, C3H/HeJ mice are immunized with 50 ug of KLH-conjugatedpeptides corresponding to OmpA₂₃₋₄₀, OmpA₄₁₋₅₈, OmpA₅₉₋₇₄, Asp14₁₀₀₋₁₁₂,Asp14₁₁₃₋₁₂₄ and every possible combination thereof. The same adjuvantsand immunization schedule as in the preceding paragraph may be followed.

Five days following the second boost, aliquots of serum from each mouseare tested via ELISA to confirm that a humoral immune response wasmounted against OmpA, Asp14, and the respective portions thereof. At oneweek following the second boost, each mouse is inoculated with either100 ul of blood from an Aph infected SCID mouse that was confirmed to beinfected or 100 ul of host cell free Aph bacteria recovered from tissuecell culture. On days 3, 8, and 12 post tick feeding, peripheral bloodis collected. DNA isolated from the blood is subjected to quantitativePCR using primers targeting the Aph 16S rDNA and murine Beta-actin todetermine the pathogen load in the peripheral blood (data not shown).

This protocol is also useful when adjuvants suitable for innocculatingdogs, humans, or other mammals are used for respective species.

In summary, OmpA and Asp14 are the first two Aph surface proteins foundto be critical for infection of mammalian cells. Expression of theseproteins is induced in Aph during the tick bloodmeal and during theperiod in which humoral immune responses are stimulated in humans andmice. Embodiments of the invention are compositions comprising OmpAand/or Asp14 sequences and methods to prevent Aph infection of humansand animals by inducing an immune response that blocks one or more ofthe 3 critical stages of infection: (1) the initial colonization ofneutrophils and/or endothelial cells that establishes infection; (2) thedissemination stage when infected peripherally circulating neutrophilsare inhibited in their microbial killing capability; and (3) theinfection of endothelial cells of heart and liver. A further embodimentprovides compositions and methods for diagnosis of anaplasmosis and HGA.

What is claimed is:
 1. A method of treating a subject from an Anaplasmaphagocytophilum infection comprising administering to said subject animmunogenic composition having at least one polypeptide comprising SEQID NO: 03 or SEQ ID NO:
 06. 2. The method according to claim 1, whereinsaid immunogenic composition comprises at least one polypeptidecomprising SEQ ID NO:03 and SEQ ID NO:06.
 3. The method of claim 1,wherein said subject is an animal and said Anaplasma phagocytophiluminfection is anaplasmosis.
 4. The method according to claim 1, whereinsaid immunogenic composition comprises at least one polypeptidecomprising SEQ ID NO:
 03. 5. The method according to claim 1, whereinsaid immunogenic composition comprises at least one polypeptidecomprising SEQ ID NO: 06.